Method for treating disorders associated with glomerular function

ABSTRACT

Methods of administering and pharmaceutical compositions of a biphenyl sulfonamide compound which is a dual angiotensin and endothelin receptor antagonist are disclosed for treating diseases.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to a biphenyl sulfonamide compound whichis a dual angiotensin and endothelin receptor antagonist, topharmaceutical compositions containing such compound, to methods ofmanufacturing the pharmaceutical formulations containing such compound,and to methods of using such compound in the treatment ofendothelin-dependent or angiotensin II-dependent disorders and otherdiseases.

Description of the Related Art

Angiotensin II (AngII) and endothelin-1 (ET-1) are two of the mostpotent endogenous vasoactive peptides currently known and are believedto play a role in controlling both vascular tone and pathological tissueremodeling associated with a variety of diseases including diabeticnephropathy, heart failure, and chronic or persistently elevated bloodpressure. Currently, angiotensin receptor blockers (ARBs), which blockthe activity of AngII, are widely used as a treatment for diabeticnephropathy, heart failure, chronic or persistently elevated bloodpressure. In addition, there is a growing body of data that demonstratesthe potential therapeutic benefits of ET receptor antagonists (ERAs) inblocking ET-1 activity.

It is also known that AngII and ET-1 work together in blood pressurecontrol and pathological tissue remodeling. For example, ARBs not onlyblock the action of AngII at its receptor, but also limit the productionof ET-1. Similarly, ERAs block ET-1 activity and inhibit the productionof AngII. Consequently, simultaneously blocking AngII and ET-1activities may offer better efficacy than blocking either substancealone.

In well-validated rat models of human chronic or persistently elevatedblood pressure, the combination of an ARB and an ERA results in asynergistic effect. Furthermore, although ARBs are the standard of carefor patients with diabetic nephropathy, improved efficacy with theco-administration of an ERA has been reported in Phase 2 clinicaldevelopment.

There are preclinical and initial clinical data suggesting that comparedto either mechanism alone, simultaneously blocking angiotensin II andendothelin 1 at their respective receptors, ATI and ETA, may provide animproved treatment option for several cardiovascular diseases.

SUMMARY OF THE INVENTION

Methods of administering, pharmaceutical dosage forms, pharmaceuticalformulations, and treatment regimes of a biphenyl sulfonamide compoundof the following formula I, enantiomers (including atropisomers),diastereomers, salts and metabolites thereof, methods of manufacturingthe pharmaceutical formulations, and methods of using the formulationsare disclosed:

Some embodiments provide a method of treating an endothelin-dependent orangiotensin II-dependent disorder in a subject in need thereof whichcomprises administering a compound of formula I or a pharmaceuticallyacceptable salt thereof, in an amount effective therefor. In some otherembodiments, the endothelin-dependent or angiotensin II-dependentdisorder is diabetic nephropathy. In some embodiments, theendothelin-dependent or angiotensin II-dependent disorder is chronicelevated blood pressure. In some embodiments, the endothelin-dependentor angiotensin II-dependent disorder is persistently elevated bloodpressure. In some embodiments, the endothelin-dependent or angiotensinII-dependent disorder is hypertension.

In some embodiments, the method comprises administering a compound offormula I or a pharmaceutically acceptable salt thereof, in an amountfrom about 50 mg/day to about 1000 mg/day. In some embodiments, theamount of the compound of formula I, or a pharmaceutically acceptablesalt thereof, administered to the human subject can be from about 200mg/day to about 800 mg/day, more preferably about 400 mg/day, mostpreferably about 800 mg/day. In some embodiments, the amount of thecompound of formula I, or a pharmaceutically acceptable salt thereof,administered to the human subject can be about 100 mg/day, about 200mg/day, about 400 mg/day, or about 800 mg/day.

In some embodiments, the systolic blood pressure of the human subject isdecreased to below at least 160 mmHg, below at least 140 mmHg, below atleast 140 mmHg, below at least 130 mmHg, or below at least 120 mmHg. Insome embodiments, the diastolic blood pressure of the human subject isdecreased to below at least 120 mmHg, below at least 110 mmHg, below atleast 100 mmHg, or below at least 90 mmHg. In some embodiments, thesystolic or diastolic blood pressure of the human subject is decreasedby at least about 5 mmHg, at least about 8 mmHg, about 10 mmHg, at leastabout 12 mmHg, or about 14 mmHg as compared to the systolic or diastolicblood pressure prior to treatment.

In some embodiments, the compound of formula I, or a pharmaceuticallyacceptable salt thereof, is administered at a frequency not greater thanfour times, twice, or once a day. In some embodiments, the compound offormula I, or a pharmaceutically acceptable salt thereof, isadministered four times, twice, or once a day.

In some embodiments, the systolic blood pressure of less than 140 mmHgor diastolic blood pressure of less than 90 mmHg is reached within 16weeks, within 14 weeks, within 12 weeks, within 10 weeks, or within 8weeks of administration of the compound of compound I, or apharmaceutically acceptable salt thereof.

Some embodiments provide a pharmaceutical composition comprising acompound of formula I, or a pharmaceutically acceptable salt thereof,for use in treating an endothelin-dependent or angiotensin II-dependentdisorder in a subject in need thereof. In some embodiments, the amountof the compound of formula I or a pharmaceutically acceptable saltthereof is from about 50 mg to about 1000 mg. In some embodiments, theendothelin-dependent or angiotensin II-dependent disorder is chronicelevated blood pressure. In some embodiments, the endothelin-dependentor angiotensin II-dependent disorder is persistently elevated bloodpressure. In some embodiments, the endothelin-dependent or angiotensinII-dependent disorder is diabetic nephropathy. In some embodiments, theendothelin-dependent or angiotensin II-dependent disorder ishypertension. In some embodiments, the amount of compound of formula I,or a pharmaceutically acceptable salt thereof, is from about 200 mg toabout 800 mg. In some embodiments, the amount of compound of formula I,or a pharmaceutically acceptable salt thereof, is about 100 mg, about200 mg, about 400 mg, or about 800 mg.

In some embodiments, the use of the pharmaceutical compositioncomprising a compound of formula I, or a pharmaceutically acceptablesalt thereof, in treating the endothelin-dependent or angiotensinII-dependent disorder is decreasing the systolic blood pressure of thehuman subject so that it is below at least 160 mmHg, below at least 150mmHg, below at least 140 mmHg, below at least 130 mmHg, or below atleast 120 mmHg. In some embodiments, the use of the pharmaceuticalcomposition comprising a compound of formula I, or a pharmaceuticallyacceptable salt thereof, in treating the endothelin-dependent orangiotensin II-dependent disorder is decreasing the diastolic bloodpressure of the human subject so that it is below at least 120 mmHg,below at least 110 mmHg, below at least 100 mmHg, or below at least 90mmHg. In some embodiments, the use of the pharmaceutical compositioncomprising a compound of formula I, or a pharmaceutically acceptablesalt thereof, in treating the endothelin-dependent or angiotensinII-dependent disorder is decreasing the systolic or diastolic bloodpressure of the human subject by at least about 5 mmHg, at least about 8mmHg, at least about 10 mmHg, at least about 12 mmHg, or at least about14 mmHg.

In some embodiments, the pharmaceutical composition of the compound offormula I, or a pharmaceutically acceptable salt thereof, isadministered at a frequency not greater than four times, twice, or oncea day. In some embodiments, the pharmaceutical composition of thecompound of formula I, or a pharmaceutically acceptable salt thereof, isadministered four times, twice, or once a day.

In some embodiments, the systolic blood pressure of less than 140 mmHgor diastolic blood pressure of less than 90 mmHg is reached within 16weeks, within 14 weeks, within 12 weeks, within 10 weeks, or within 8weeks of administration of the pharmaceutical composition of thecompound of compound I, or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula I is provided as rapidlydissolving dosage forms. In some embodiments, the formulations have oneor more of: improved friability, compression, dissolution, uniformity,dissolvability, palatability, and the like. Also, in some embodiments,the formulations can permit at least one or more of: rapid onset,greater and/or more rapid plasma levels, and the like.

Some embodiments provide a pharmaceutical composition comprising fromabout 100 mg to about 800 mg of the compound of formula I or apharmaceutically acceptable salt thereof, and at least one excipient,wherein the compound of formula I comprises greater than about 35% w/wof the composition and the excipients comprise from about 5% to about65% w/w of the composition. In some embodiments, each excipient isindividually selected from the group consisting of microcrystallinecellulose, hydroxypropylcellulose, poloxamer 188, sodium starchglycolate, and croscarmellose sodium. In some embodiments, the compoundof formula I is provided in an amount from about 40% to about 65% w/w ofthe composition, or about 50% to about 60% w/w of the composition. Insome embodiments, one excipient is microcrystalline cellulose. In someembodiments, the microcrystalline cellulose can comprise from about 5%to about 65% w/w, from about 15% to about 50% w/w, from about 20% toabout 40% w/w, from about 25% to about 35% w/w, or about 28% to about30% w/w of the composition. In some embodiments, the microcrystallinecellulose is silicified microcrystalline cellulose. In some embodiments,one excipient is poloxamer 188. In some embodiments, poloxamer 188comprises from about 0.1% to about 10% w/w, or from about 1% to about 8%w/w of the composition. In some embodiments, poloxamer 188 comprisesabout 5% w/w of the composition.

In some embodiments, the composition further comprises one or morelubricants, wherein the one or more lubricants comprise up to about 5%w/w of the composition. In some embodiments, the one or more lubricantsare each individually selected from the group consisting of sodiumlauryl sulfate, magnesium stearate, calcium stearate, sodium stearylfumarate, stearic acid, hydrogenated vegetable oil, glyceryl behenate,and polyethylene glycol. In some embodiments, one lubricant is magnesiumstearate. In some embodiments, magnesium stearate comprises from about0.1% to about 1.5% w/w of the composition. In some embodiments,magnesium stearate comprises about 0.5% w/w or about 1.0% w/w of thecomposition. In some embodiments, one lubricant is sodium laurylsulfate. In some embodiments, sodium lauryl sulfate comprises from about0.1% to about 5% w/w, or from about 0.3% to about 2% w/w of thecomposition. In some embodiments, sodium lauryl sulfate comprises about1.0% w/w of the composition. In some embodiments, the compositionfurther comprises a glidant. In some embodiments, the glidant iscolloidal silicon dioxide. In some embodiments, the colloidal silicondioxide comprises about 0.01% to about 1.5% w/w of the composition. Insome embodiments, the colloidal silicon dioxide comprises about 0.1% w/wof the composition. In some embodiments, the compound of formula I isprovided in an amount of about 100 mg, or about 200 mg, or about 400 mg,or about 800 mg.

Some embodiments provide a pharmaceutical composition comprising fromabout 100 mg to about 800 mg of the compound of formula I, or apharmaceutically acceptable salt thereof, and at least one excipient. Insome embodiments, the excipient is selected from the group consisting ofmicrocrystalline cellulose, lactose monohydrate, croscarmellose sodium,sodium starch glycolate, hydroxypropyl cellulose, and poloxamer 188. Insome embodiments, one excipient is microcrystalline cellulose whichcomprises from about 20% to about 50% w/w, or from about 20% to about40% w/w, or from about 28% to about 30% w/w of the composition. In someembodiments, the composition further comprises a glidant. In someembodiments, the composition further comprises one or more lubricants.In some embodiments, the composition further comprises one or moresurfactants.

In some embodiments, the compound of formula I is provided in an amountof about 100 mg, or about 200 mg, or about 400 mg, or about 800 mg.

In some embodiments, the composition has a total weight of about 50 mgto about 1500 mg. In some embodiments, the composition has a totalweight of about 50 mg, about 75 mg, about 100 mg, about 150 mg, about175 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about700 mg, about 750 mg, about 800 mg, about 900 mg, about 1000 mg, about1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, or about 1500 mg.

In some embodiments, the composition is in the form of a tablet, a filmcoated tablet, a capsule, a gel cap, a caplet, a pellet, or a bead. Insome embodiments, the composition is in the form of a film coatedtablet.

Some embodiments provides a tablet comprising from about 100 mg to about800 mg the compound of formula I or a pharmaceutically acceptable saltthereof, the tablets having a hardness of at least about 4 Kp. In someembodiments, the tablet has a hardless of at least about 6 Kp, or atleast about 8 Kp, or at least about 10 Kp, or at least about 12 Kp, orat least about 14 Kp. In some embodiments, the tablet has a hardness ofabout 4 Kp, about 6 Kp, about 8 Kp, about 10 Kp, about 12 Kp, about 14Kp, or about 16 Kp.

Some embodiments provide a pharmaceutical composition comprising fromabout 100 mg to about 800 mg of the compound of formula I, or apharmaceutically acceptable salt thereof, wherein the composition havingat least an 85 percent release of the compound of formula I, or the saltof the compound of formula I, within 45 minutes using U.S. Pharmacopeia(USP) type II dissolution Apparatus I at 50 rpm or 60 rpm in 0.1N HCl.In some embodiments, the composition has at least an 85 percent releaserate at 30 minutes, or at 20 minutes, or at 15 minutes. In someembodiments, the composition has at least a 90 percent release rate at30 minutes. In some embodiments, the composition has at least a 95percent release rate at 30 minutes.

Some embodiments provide a method of treating chronic or persistentlyelevated blood pressure, comprising identifying an individual in need ofsuch treatment, and administering the pharmaceutical compositions of thecompound of formula I.

Some embodiments provide a method of making a pharmaceutical compositioncomprising blending together from about 100 mg to about 800 mg of thecompound of formula I, and one or more excipients, wherein the one ormore excipients comprise from about 5% to about 65% w/w of thecomposition. In some embodiments, one excipient is microcrystallinecellulose. In some embodiments, the microcrystalline cellulose comprisesfrom about 20% to about 50% w/w, or from about 28% to about 30% w/w ofthe composition. In some embodiments, the method further comprisesadding from about 0.5% to about 1.0% w/w magnesium stearate. In someembodiments, the method further comprises adding about 5% w/w poloxamer188. In some embodiments, the method further comprises adding about 1.0%w/w sodium lauryl sulfate. In some embodiments, the method furthercomprises adding about 0.1% w/w colloidal silicon dioxide. In someembodiments, the compound of formula I is provided in an amount of about100 mg, or about 200 mg, or about 400 mg, or about 800 mg.

Some embodiments provide a pharmaceutical composition comprising fromabout 100 mg to about 800 mg of the compound of formula I, or apharmaceutically acceptable salt thereof, and microcrystalline cellulosewhich comprises about 29% w/w of the composition, croscarmellose sodiumwhich comprises about 5% w/w of the composition, hydroxypropylcellulosewhich comprises about 3% w/w of the composition, poloxamer 188 whichcomprises about 5% w/w of the composition, colloidal silicone dioxidewhich comprises about 0.1% w/w of the composition, and magnesiumstearate which comprises about 0.5% w/w of the composition.

Some embodiments provide a pharmaceutical composition comprising fromabout 100 mg to about 800 mg of the compound of formula I, or apharmaceutically acceptable salt thereof, and microcrystalline cellulosewhich comprises about 22% w/w of the composition, lactose monohydratewhich comprises about 11% w/w of the composition, sodium lauryl sulfatewhich comprises about 1% w/w of the composition, croscarmellose sodiumwhich comprises about 5% w/w of the composition, hydroxypropylcellulosewhich comprises about 3.0% w/w of the composition, colloidal siliconedioxide which comprises about 0.1% w/w of the composition, and magnesiumstearate which comprises about 1% w/w of the composition.

Some embodiments provide a pharmaceutical composition comprising fromabout 100 mg to about 800 mg of the compound of formula I, or apharmaceutically acceptable salt thereof, and microcrystalline cellulosewhich comprises about 29% w/w of the composition, croscarmellose sodiumwhich comprises about 5% w/w of the composition, hydroxypropylcellulosewhich comprises about 3% w/w of the composition, poloxamer 188 comprisesabout 5% w/w of the composition, colloidal silicone dioxide whichcomprises about 0.1% w/w of the composition, and magnesium stearatewhich comprises about 1% w/w of the composition.

Some embodiments provide a pharmaceutical composition comprising fromabout 100 mg to about 800 mg of the compound of formula I, or apharmaceutically acceptable salt thereof, and microcrystalline cellulosewhich comprises about 33% w/w of the composition, sodium lauryl sulfatewhich comprises about 1% w/w of the composition, croscarmellose sodiumwhich comprises about 5% w/w of the composition, hydroxypropylcellulosewhich comprises about 3% w/w of the composition, colloidal siliconedioxide which comprises about 0.1% w/w of the composition, and magnesiumstearate which comprises about 1% w/w of the composition.

Some embodiments provide a pharmaceutical composition comprising fromabout 100 mg to about 800 mg of the compound of formula I, or apharmaceutically acceptable salt thereof, and microcrystalline cellulosewhich comprises about 29% w/w of the composition, hydroxypropylcellulosewhich comprises about 3% w/w of the composition, poloxamer 188 whichcomprises about 5% w/w of the composition, sodium starch glycolate whichcomprises about 5% w/w of the composition, colloidal silicone dioxidewhich comprises about 0.1% w/w of the composition, and magnesiumstearate which comprises about 1% w/w of the composition.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The following are definitions of terms used in this specification. Theinitial definition provided for a group or term herein applies to thatgroup or term throughout the present specification, individually or aspart of another group, unless otherwise indicated.

The compound of formula I form salts which are also within the scope ofthis disclosure. Reference to a compound of formula I herein isunderstood to include reference to salts thereof, unless otherwiseindicated. The term “salt(s)”, as employed herein, denotes acidic and/orbasic salts formed with inorganic and/or organic acids and bases. Inaddition, when the compound of formula I contains both a basic moietyand an acidic moiety, zwitterions (“inner salts”) may be formed and areincluded within the term “salt(s)” as used herein. Pharmaceuticallyacceptable (i.e., non-toxic, physiologically acceptable) salts arepreferred, although other salts are also useful, e.g., in isolation orpurification steps which may be employed during preparation. Salts ofthe compound of formula I may be formed, for example, by reacting thecompound of formula I with an amount of acid or base, such as anequivalent amount, in a medium such as one in which the saltprecipitates or in an aqueous medium followed by lyophilization.

The compound of formula I which contains a basic moiety may form saltswith a variety of organic and inorganic acids. Exemplary acid additionsalts include acetates (such as those formed with acetic acid ortrihaloacetic acid, for example, trifluoroacetic acid), adipates,alginates, ascorbates, aspartates, benzoates, benzenesulfonates,bisulfates, borates, butyrates, citrates, camphorates,camphorsulfonates, cyclopentanepropionates, digluconates,dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates,glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides(formed with hydrochloric acid), hydrobromides (formed with hydrogenbromide), hydroiodides, 2-hydroxyethanesulfonates, lactates, maleates(formed with maleic acid), methanesulfonates (formed withmethanesulfonic acid), 2-naphthalenesulfonates, nicotinates, nitrates,oxalates, pectinates, persulfates, 3-phenylpropionates, phosphates,picrates, pivalates, propionates, salicylates, succinates, sulfates(such as those formed with sulfuric acid), sulfonates (such as thosementioned herein), tartrates, thiocyanates, toluenesulfonates such astosylates, undecanoates, and the like.

The compound of formula I which contains an acidic moiety may form saltswith a variety of organic and inorganic bases. Exemplary basic saltsinclude ammonium salts, alkali metal salts such as sodium, lithium, andpotassium salts, alkaline earth metal salts such as calcium andmagnesium salts, salts with organic bases (for example, organic amines)such as benzathines, dicyclohexylamines, hydrabamines (formed withN,N-bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamines,N-methyl-D-glucamides, t-butyl amines, and salts with amino acids suchas arginine, lysine and the like. Basic nitrogen-containing groups maybe quaternized with agents such as lower alkyl halides (e.g. methyl,ethyl, propyl, and butyl chlorides, bromides and iodides), dialkylsulfates (e.g. dimethyl, diethyl, dibutyl, and diamyl sulfates), longchain halides (e.g. decyl, lauryl, myristyl and stearyl chlorides,bromides and iodides), aralkyl halides (e.g. benzyl and phenethylbromides), and others.

Prodrugs and solvates of the compound of formula I are alsocontemplated. The term “prodrug” denotes a compound which, uponadministration to a subject, undergoes chemical conversion by metabolicor chemical processes to yield a compound of formula I, or a salt and/orsolvate thereof. Solvates of the compound of formula I are preferablyhydrates. Any tautomers are also contemplated.

All stereoisomers of the compound of formula I, such as those which mayexist due to asymmetric carbons on the R substituents, includingenantiomeric forms (which may exist even in the absence of asymmetriccarbons, e.g., atropisomers) and diastereomeric forms, are contemplated.Individual stereoisomers of the compound of formula I may, for example,be substantially free of other isomers, or may be admixed, for example,as racemates or with all other, or other selected, stereoisomers. Thechiral centers of the compound of formula I can have the S or Rconfiguration.

Methods of Preparation

The compound of formula I may be prepared by methods such as thoseillustrated in the following Schemes I to II. Solvents, temperatures,pressures, and other reaction conditions may be selected by one ofordinary skill in the art.

In one embodiment, the compound of formula I can be synthesized by themethod disclosed in Scheme I. Substituted 4-bromobenzonitrile formulaI-1 (X is bromine or mesylate) can be treated sodium ethoxide in DMF,for example ethyl alcohol in DMF can be treated with sodium hydride, toprovide 4-bromo-3-(ethoxymethyl)benzonitrile.4-Bromo-3-(ethoxymethyl)benzonitrile can be converted to4-bromo-3-(ethoxymethyl)benzaldehyde by reduction of the nitrile to analdehyde. For example, 4-bromo-3-(ethoxymethyl)benzonitrile can betreated with DIBAL-H followed by methanol and hydrochloric acid toprovide 4-bromo-3-(ethoxymethyl)benzaldehyde. The4-bromo-3-(ethoxymethyl)benzaldehyde can then be coupled with a compoundof formula I-2 in the presence of a palladium catalyst, for exampletetrakis (triphenylphosphine) palladium(0), under the appropriateconditions to provide a compound of formula I-3 (R is SEM or MEM). Thealdehyde of the compound of formula I-3 can be reduced to an alcoholthereby providing a compound of formula I-4. For example, the compoundof formula I-3 can be treated with sodium borohydride in ethyl alcoholor methyl alcohol to provide the compound of formula I-4 (R is SEM orMEM). The benzyl alcohol of formula I-4 can then be converted to abenzyl bromide thereby providing a compound of formula I-5 (R is SEM orMEM). For example, benzyl alcohol of formula I-4 in DMF in the presenceof carbon tetrabromide can be treated with triphenyl phosphine toprovide benzyl bromide of formula I-5. The benzyl bromide of formula I-5can be treated with 2-N-butyl-1,3-diazaspiro[4.4]non-1-en-4-onehydrochloride to provide the compound of formula I-6 (R is SEM or MEM).For example, 2-N-butyl-1,3-diazaspiro[4.4]non-1-en-4-one hydrochloridecan be treated with sodium hydride in DMF followed by addition of thebenzyl bromide of formula I-5 to provide the compound of formula I-6.The compound of formula I-6 can be deprotected under acidic conditions,for example the compound of formula I-6 in ethyl alcohol can be treatedwith 6N hydrochloric acid, thereby providing the compound of formula I,4′-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-N-(4,5-dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)[1,1′-biphenyl]-2-sulfonamide.

In another embodiment, the compound of formula I can be synthesized bythe method disclosed in Scheme II. Ethyl 4-bromo-3-methylbenzoate (110g, 450 mmol) can be treated with NBS to provide ethyl4-bromo-3-methylbenzoate. Ethyl 4-bromo-3-methylbenzoate can be treatedsodium ethoxide in ethyl alcohol, for example a 21% of sodium ethoxidesolution in ethanol, to provide ethyl 4-bromo-3-(ethoxymethyl)benzoate.Ethyl 4-bromo-3-(ethoxymethyl)benzoate can then be coupled with2-[[N-(4,5-dimethyl-3-isoxazolyl)-N-(methoxymethyl)amino]sulfonyl]-phenylboronicacid in the presence of a palladium catalyst, for example tetrakis(triphenylphosphine) palladium(0), under the appropriate conditions toprovideN-(4,5-dimethyl-3-isoxazolyl)-4′-(ethoxycarbonyl)-2′-(ethoxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamide.N-(4,5-Dimethyl-3-isoxazolyl)-4′-(ethoxycarbonyl)-2′-(ethoxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamidecan be reduced to an alcohol thereby providingN-(4,5-Dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)-4′-(hydroxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamide.For example,N-(4,5-dimethyl-3-isoxazolyl)-4′-(ethoxycarbonyl)-2′-(ethoxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamide in THF can be treated with a solution ofDIBAL-H in toluene to provideN-(4,5-dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)-4′-(hydroxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamide.N-(4,5-Dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)-4′-(hydroxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamidecan then be converted to4′-(bromomethyl)-N-(4,5-dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamide.For example,N-(4,5-dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)-4′-(hydroxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamidein DMF in the presence of carbon tetrabromide can be treated withbiphenyl phosphine to provide4′-(bromomethyl)-N-(4,5-dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamide.4′-(Bromomethyl)-N-(4,5-dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamidecan be treated with 2-N-butyl-1,3-diazaspiro[4.4]non-1-en-4-onehydrochloride to provide4′-[(2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl-N-](4,5-dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamide.For example, 2-N-butyl-1,3-diazaspiro[4.4]non-1-en-4-one hydrochloridecan be treated with sodium hydride in DMF followed by addition of4′-(bromomethyl)-N-(4,5-dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamideto provide4′-[(2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl-N-](4,5-dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamide.4′-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl-N-](4,5-dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamidecan be deprotected under acidic conditions, thereby providing thecompound of formula I,4′-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-N-(4,5-dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)[1,1′-biphenyl]-2-sulfonamide.For example4′-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl-N-](4,5-dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamidein ethyl alcohol can be treated with 6N hydrochloric acid to provide thecompound of formula I,4′-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-N-(4,5-dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)[1,1′-biphenyl]-2-sulfonamide.

The compound of formula I and salts thereof are antagonists of bothendothelin (especially, ET-1) and angiotensin II (especially, subtypeAT₁) receptors (“dual angiotensin endothelin receptor antagonists”) andare useful in treatment of conditions associated with increased ETlevels and/or increased angiotensin II levels and of allendothelin-dependent or angiotensin II-dependent disorders. They arethus useful as antihypertensive agents. By the administration of acomposition having the compound of formula I, the blood pressure of ahypertensive mammalian (e.g., human) host is reduced. They are alsouseful in portal chronic or persistently elevated blood pressure,chronic or persistently elevated blood pressure secondary to treatmentwith erythropoietin, low renin chronic or persistently elevated bloodpressure, and chronic or persistently elevated blood pressure.

The compound of formula I is also useful in the treatment of disordersrelated to renal, glomerular and mesangial cell function, includingacute (such as ischemic, nephrotoxic, or glomerulonephritis) and chronic(such as diabetic, hypertensive or immune-mediated) renal failure,diabetic nephropathy, glomerular injury, renal damage secondary to oldage or related to dialysis, nephrosclerosis (especially hypertensivenephrosclerosis), nephrotoxicity (including nephrotoxicity related toimaging and contrast agents and to cyclosporine), renal ischemia,primary vesicoureteral reflux, glomerulosclerosis and the like. Thecompound of formula I is also useful in the treatment of disordersrelated to paracrine and endocrine function. The compound of formula Iis also useful in the treatment of diabetic nephropathy, IGA-inducednephropathy, and hypertension-induced nephropathy. The term “diabeticnephropathy” as used herein will be understood to include both incipientand overt stages of diabetic nephropathy, whether diagnosed or not, butmost typically as diagnosed by a clinician or physician.

The compound of formula I is also useful in the treatment of endotoxemiaor endotoxin shock as well as hemorrhagic shock. The compound of formulaI is also useful in alleviation of pain associated cancer, such as thepain associated with prostate cancer, and bone pain associated with bonecancer. The compound of formula I is further useful in the preventionand/or reduction of end-organ damage associated the cell-proliferativeeffects of endothelin.

The compound of formula I is also useful in hypoxic and ischemic diseaseand as anti-ischemic agents for the treatment of, for example, cardiac,renal and cerebral ischemia and reperfusion (such as that occurringfollowing cardiopulmonary bypass surgery), coronary and cerebralvasospasm, and the like.

In addition, the compound of formula I is also useful as anti-arrhythmicagents; anti-anginal agents; anti-fibrillatory agents; anti-asthmaticagents; anti-atherosclerotic and anti-arteriosclerotic agents (includinganti-transplantation arteriosclerotic agents); additives to cardioplegicsolutions for cardiopulmonary bypasses; adjuncts to thrombolytictherapy; and anti-diarrheal agents. The compound of formula I may beuseful in therapy for myocardial infarction; therapy for peripheralvascular disease (e.g., Raynaud's disease, intermittent claudication andTakayashu's disease); treatment of cardiac hypertrophy (e.g.,hypertrophic cardiomyopathy); treatment of primary pulmonaryhypertension (e.g., plexogenic, embolic) in adults and in the newbornand pulmonary hypertension secondary to heart failure, radiation andchemotherapeutic injury, or other trauma; treatment of central nervoussystem vascular disorders, such as stroke, migraine and subarachnoidhemorrhage; treatment of central nervous system behavioral disorders;treatment of gastrointestinal diseases such as ulcerative colitis,Crohn's disease, gastric mucosal damage, ulcer, inflammatory boweldisease and ischemic bowel disease; treatment of gall bladder or bileduct-based diseases such as cholangitis; treatment of pancreatitis;regulation of cell growth; treatment of benign prostatic hypertrophy;restenosis following angioplasty or following any procedure includingtransplantation and stenting; therapy for congestive heart failureincluding inhibition of fibrosis; inhibition of left ventriculardilatation, remodeling and dysfunction; and treatment of hepatotoxicityand sudden death. The compound of formula I is useful in the treatmentof sickle cell disease including the initiation and/or evolution of thepain crises of this disease; treatment of the deleterious consequencesof ET-producing tumors such as chronic or persistently elevated bloodpressure resulting from hemangiopericytoma; treatment of early andadvanced liver disease and injury including attendant complications(e.g., hepatotoxicity, fibrosis and cirrhosis); treatment of spasticdiseases of the urinary tract and/or bladder; treatment of hepatorenalsyndrome; treatment of immunological diseases involving vasculitis suchas lupus, systemic sclerosis, mixed cryoglobulinemia; and treatment offibrosis associated with renal dysfunction and hepatotoxicity. Thecompound of formula I is useful in therapy for metabolic andneurological disorders; cancer; insulin-dependent and noninsulin-dependent diabetes mellitus; neuropathy; retinopathy; epilepsy;hemorrhagic and ischemic stroke; bone remodeling; psoriasis; and chronicinflammatory diseases such as arthritis, rheumatoid arthritis,osteoarthritis, sarcoidosis and eczematous dermatitis (all types ofdermatitis).

The compound of formula I is additionally useful in the treatment ofdisorders involving bronchoconstriction and disorders of chronic oracute pulmonary inflammation such as chronic obstructive pulmonarydisease (COPD) and adult respiratory distress syndrome (ARDS).

The compound of formula I is also useful in the treatment of sexualdysfunction in both men (erectile dysfunction, for example, due todiabetes mellitus, spinal cord injury, radical prostatectomy,psychogenic etiology or any other cause) and women by improving bloodflow to the genitalia, especially, the corpus cavernosum.

The compound of formula I is also useful in the treatment of dementia,including Alzheimer's dementia, senile dementia and vascular dementia.Additionally, the compound of formula I is further useful in thereduction of general morbidity and/or mortality as a result of the aboveutilities. Methods for the treatment of all endothelin-dependent orangiotensin II-dependent disorders, comprising the step of administeringto a subject in need thereof the compound of formula I in an amounteffective therefore are provided. Other therapeutic agents such as thosedescribed below may be employed with the compound of formula I in thepresent methods. In the methods, such other therapeutic agent(s) may beadministered prior to, simultaneously with or following theadministration of the compound of formula I of the present disclosure.

Pharmaceutical Compositions

In another aspect, the present disclosure relates to a pharmaceuticalcomposition comprising a physiologically acceptable surface activeagents, carriers, diluents, excipients, smoothing agents, suspensionagents, film forming substances, and coating assistants, or acombination thereof; and a compound disclosed herein. Acceptablecarriers or diluents for therapeutic use are well known in thepharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa.(1990), which is incorporated herein by reference in its entirety.Preservatives, stabilizers, dyes, sweeteners, fragrances, flavoringagents, and the like may be provided in the pharmaceutical composition.For example, sodium benzoate, ascorbic acid and esters ofp-hydroxybenzoic acid may be added as preservatives. In addition,antioxidants and suspending agents may be used. In various embodiments,alcohols, esters, sulfated aliphatic alcohols, and the like may be usedas surface active agents; sucrose, glucose, lactose, starch,crystallized cellulose, mannitol, light anhydrous silicate, magnesiumaluminate, magnesium methasilicate aluminate, synthetic aluminumsilicate, calcium carbonate, sodium acid carbonate, calcium hydrogenphosphate, calcium carboxymethyl cellulose, and the like may be used asexcipients; magnesium stearate, talc, hardened oil and the like may beused as smoothing agents; coconut oil, olive oil, sesame oil, peanutoil, soya may be used as suspension agents or lubricants; celluloseacetate phthalate as a derivative of a carbohydrate such as cellulose orsugar, or methylacetate-methacrylate copolymer as a derivative ofpolyvinyl may be used as suspension agents; and plasticizers such asester phthalates and the like may be used as suspension agents.

The term “pharmaceutical composition” refers to a mixture of a compounddisclosed herein with other chemical components, such as diluents orcarriers. The pharmaceutical composition facilitates administration ofthe compound to an organism. Multiple techniques of administering acompound exist in the art including, but not limited to, oral,injection, aerosol, parenteral, and topical administration.Pharmaceutical compositions can also be obtained by reacting compoundswith inorganic or organic acids such as hydrochloric acid, hydrobromicacid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and thelike.

The term “carrier” defines a chemical compound that facilitates theincorporation of a compound into cells or tissues. For example dimethylsulfoxide (DMSO) is a commonly utilized carrier as it facilitates theuptake of many organic compounds into the cells or tissues of anorganism.

The term “diluent” defines chemical compounds diluted in water that willdissolve the compound of interest as well as stabilize the biologicallyactive form of the compound. Salts dissolved in buffered solutions areutilized as diluents in the art. One commonly used buffered solution isphosphate buffered saline because it mimics the salt conditions of humanblood. Since buffer salts can control the pH of a solution at lowconcentrations, a buffered diluent rarely modifies the biologicalactivity of a compound.

The term “physiologically acceptable” defines a carrier or diluent thatdoes not abrogate the biological activity and properties of thecompound.

The pharmaceutical compositions described herein can be administered toa human patient per se, or in pharmaceutical compositions where they aremixed with other active ingredients, as in combination therapy, orsuitable carriers or excipient(s). Techniques for formulation andadministration of the compound of formula I may be found in “Remington'sPharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 18thedition, 1990.

Some embodiments provide low-dose the compound of formula I in tablets,film coated tablets, capsules, caplets, pills, gel caps, pellets, beads,or dragee dosage forms. Preferably, the formulations disclosed hereincan provide favorable drug processing qualities, including, for example,but not limited to, rapid tablet press speeds, reduced compressionforce, reduced ejection forces, blend uniformity, content uniformity,uniform dispersal of color, accelerated disintegration time, rapiddissolution, low friability (preferable for downstream processing suchas packaging, shipping, pick-and-pack, etc.) and dosage form physicalcharacteristics (e.g., weight, hardness, thickness, friability) withlittle variation.

In other embodiments, the formulation yields a rapidly dissolving dosageform, for which at least 85% of the labeled amount of the drug substancedissolves within 45 minutes, using U.S. Pharmacopeia (USP) type IIdissolution Apparatus utilizing 0.1N HCl at 37° C. with 50 rpm paddlespeed. In other embodiments, the formulation yields a rapidly dissolvingdosage form, for which at least 85% of the labeled amount of the drugsubstance dissolves within 45 minutes, using U.S. Pharmacopeia (USP)type II dissolution Apparatus utilizing 0.1N HCl at 37° C. with 60 rpmpaddle speed. In other embodiments, the formulation yields a rapidlydissolving dosage form, for which at least 85% of the labeled amount ofthe drug substance dissolves within 30 minutes, using U.S. Pharmacopeia(USP) type II dissolution Apparatus utilizing 0.1N HCl at 37° C. with 50rpm paddle speed. In other embodiments, the formulation yields a rapidlydissolving dosage form, for which at least 85% of the labeled amount ofthe drug substance dissolves within 30 minutes, using U.S. Pharmacopeia(USP) type II dissolution Apparatus utilizing 0.1N HCl at 37° C. with 50rpm paddle speed. In other embodiments, the formulation yields a rapidlydissolving dosage form, for which at least 85% of the labeled amount ofthe drug substance dissolves within 20 minutes, using U.S. Pharmacopeia(USP) type II dissolution Apparatus utilizing 0.1N HCl at 37° C. with 50rpm paddle speed. In other embodiments, the formulation yields a rapidlydissolving dosage form, for which at least 85% of the labeled amount ofthe drug substance dissolves within 20 minutes, using U.S. Pharmacopeia(USP) type II dissolution Apparatus utilizing 0.1N HCl at 37° C. with 50rpm paddle speed.

In some embodiments, the formulations require minimal tablet compressionforces to achieve a hardness of about 2 kp to about 25 kp. In someaspects, the formulation can require compression forces to achieve ahardness of, for example, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 kp. Such minimalcompression forces can enables the tablets to remain relatively porousand disintegrate fast with minimal wear on compression tooling and thetablet press.

In other embodiments, the formulations can yield tablets, including filmcoated tablets, having a friability value of 1% or less. Thus, in someembodiments, the friability value is about 0.9%, 0.8%, 0.75%, 0.6%,0.5%, 0.4%, 0.3%, 0.25%, 0.2%, 0.15%, 0.1%, 0.08%, 0.06%, 0.04%, 0.02%or less.

The compound of formula I can be formulated readily, for example, bycombining the drug substance with any suitable pharmaceuticallyacceptable excipient(s) for example, but not limited to, binders,diluents, disintegrants, lubricants, fillers, carriers, coatings,glidants, flavours, color additives, and the like, as set forth below.Such compositions can be prepared for storage and for subsequentprocessing.

Excipients

Acceptable excipients for therapeutic use are well known in thepharmaceutical art, and are described, for example, in Handbook ofPharmaceutical Excipients, 5th edition (Raymond C Rowe, Paul J Sheskeyand Sian C Owen, eds. 2005), and Remington: The Science and Practice ofPharmacy, 21st edition (Lippincott Williams & Wilkins, 2005), each ofwhich is hereby incorporated in its entirety. The term “carrier”material or “excipient” herein can mean any substance, not itself atherapeutic agent, used as a carrier and/or diluent and/or adjuvant, orvehicle for delivery of a therapeutic agent to a subject or added to apharmaceutical composition to improve its handling or storage propertiesor to permit or facilitate formation of a dose unit of the compositioninto a discrete article such as a capsule, tablet, film coated tablet,caplet, gel cap, pill, pellet, bead, and the like suitable for oraladministration. Excipients can include, by way of illustration and notlimitation, diluents, disintegrants, binding agents, wetting agents,polymers, lubricants, glidants, coatings, sweetens, solubilizing agenssubstances added to mask or counteract a disagreeable taste or odor,flavors, colorants, fragrances, and substances added to improveappearance of the composition.

Acceptable excipients include, for example, but are not limited to,microcrystalline cellulose, lactose, sucrose, starch powder, maizestarch or derivatives thereof, cellulose esters of alkanoic acids,cellulose alkyl esters, talc, stearic acid, magnesium stearate,magnesium oxide, sodium and calcium salts of phosphoric and sulfuricacids, gelatin, acacia gum, sodium alginate, polyvinyl-pyrrolidone,and/or polyvinyl alcohol, saline, dextrose, mannitol, lactosemonohydrate, lecithin, albumin, sodium glutamate, cysteinehydrochloride, croscarmellose sodium, sodium starch glycolate,hydroxypropyl cellulose, poloxamer (e.g., poloxamers 101, 105, 108, 122,123, 124, 181, 182, 183, 184, 185, 188, 212, 215, 217, 231, 234, 235,237, 238, 282, 284, 288, 331, 333, 334, 335, 338, 401, 402, 403, 407,and poloxamer 105 benzoate, poloxamer 182 dibenzoate 407, and the like),sodium lauryl sulfate, colloidal silicon dioxide and the like. Examplesof suitable excipients for tablets and capsules include, but are notlimited to, microcrystalline cellulose, silicified microcrystallinecellulose, lactose monohydrate, croscarmellose sodium, sodium starch,hydroxypropyl cellulse, poloxamer 188, sodium lauryl sulfate, colloidalsilicon dioxide, magnesium stearate. Examples of suitable excipients forsoft gelatin capsules include vegetable oils, waxes, fats, semisolid andliquid polyols. Suitable excipients for the preparation of solutions andsyrups include, without limitation, water, polyols, sucrose, invertsugar and glucose. The compound can also be made in microencapsulatedform. If desired, absorption enhancing preparations (for example,liposomes), can be utilized.

The compositions and formulations can include any other agents thatprovide improved transfer, delivery, tolerance, and the like. Thesecompositions and formulations can include, for example, powders, pastes,jellies, waxes, oils, lipids, lipid (cationic or anionic) containingvesicles (such as Lipofectin™), DNA conjugates, anhydrous absorptionpastes, oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax.

Any of the foregoing mixtures can be appropriate in treatments andtherapies in accordance with the disclosure herein, provided that theactive ingredient in the formulation is not inactivated by theformulation and the formulation is physiologically compatible andtolerable with the route of administration. See also Baldrick P.“Pharmaceutical excipient development: the need for preclinicalguidance.” Regul. Toxicol. Pharmacol. 32(2):210-8 (2000), Charman W N“Lipids, lipophilic drugs, and oral drug delivery-some emergingconcepts.” J. Pharm. Sci. 89(8):967-78 (2000), and the citations thereinfor additional information related to formulations, excipients andcarriers well known to pharmaceutical chemists.

In some embodiments, one or more, or any combination of the listedexcipients can be specifically included or excluded from theformulations and/or methods disclosed herein.

As will be appreciated by those of skill in the art, the amounts ofexcipients will be determined by drug dosage and dosage form size. Insome embodiments disclosed herein, the dosage form size is about 175 mg.In some embodiment disclosed here in the dosage form size is about 350mg. In some embodiment disclosed here in the dosage form size is about700 mg. This dosage form weight is arbitrary and one skilled in the artwill realize that a range of weights can be made and are encompassed bythis disclosure. The preferred dosage form range is from about 50 mg toabout 1500 mg, more typically from about 100 mg to about 1000 mg, moretypically from about 175 mg to abpit 700 mg, with the preferred typicalform weight being about 175 mg, about 350 mg, or about 700 mg.

Lubricants

In some embodiments, lubricants are employed in the manufacture ofcertain dosage forms. For example, a lubricant will often be employedwhen producing tablets. In some embodiments, a lubricant can be addedjust before the tableting step, and can be mixed with the formulationfor a minimum period of time to obtain good dispersal. In someembodiments, one or more lubricants can be used. Examples of suitablelubricants include, but are not limited to, magnesium stearate, calciumstearate, zinc stearate, stearic acid, talc, glyceryl behenate,polyethylene glycol, polyethylene oxide polymers (for example, availableunder the registered trademarks of Carbowax® for polyethylene glycol andPolyox® for polyethylene oxide from Dow Chemical Company, Midland,Mich.), sodium lauryl sulfate, magnesium lauryl sulfate, sodium oleate,sodium stearyl fumarate, DL-leucine, colloidal silica, and others asknown in the art. Typical lubricants are magnesium stearate, calciumstearate, zinc stearate and mixtures of magnesium stearate with sodiumlauryl sulfate. Lubricants can comprise from about 0.25% to about 50% ofthe tablet weight, typically from about 1% to about 40%, more typicallyfrom about 5% to about 30%, most typically from 20% to 30%. In someembodiments, magnesium stearate can be added as a lubricant, forexample, to improve powder flow, prevent the blend from adhering totableting equipment and punch surfaces and provide lubrication to allowtablets to be cleanly ejected from tablet dies. Magnesium stearate cantypically be added to pharmaceutical formulations at concentrationsranging from about 0.1% to about 5.0% w/w, or from about 0.25% to about4% w/w, or from about 0.5% % to about 3% w/w, or from about 0.75% toabout 2% w/w, or from about 0.8% to about 1.5% w/w, or from about 0.85%to about 1.25% w/w, or from about 0.9% to about 1.20% w/w, or from about0.85% to about 1.15% w/w, or from about 0.90% to about 1.1.% w/w, orfrom about 0.95% to about 1.05% w/w, or from about 0.95% to about 1%w/w. The above ranges are examples of typical ranges. One of ordinaryskill in the art would recognize additional lubricants and/or amountsthat can be used in the formulations described herein. As would berecognized by one of ordinary skill in the art, when incorporated intothe formulations disclosed herein, the amounts of the major filler(s)and/or other excipients can be reduced accordingly to accommodate theamount of lubricant(s) added in order to keep the overall unit weight ofthe tablet unchanged.

Color Additives

In some embodiments, color additives also can be included. The colorantscan be used in amounts sufficient to distinguish dosage form strengths.Preferably, color additives approved for use in drugs (21 CFR 74, whichis incorporated herein by reference in its entirety) are added to thecommercial formulations to differentiate tablet strengths. The use ofother pharmaceutically acceptable colorants and combinations thereof areencompassed by the current disclosure.

Binders

Binders can be used, for example, to impart cohesive qualities to aformulation, and thus ensure that the resulting dosage form remainsintact after compaction. Suitable binder materials include, but are notlimited to, microcrystalline cellulose, gelatin, sugars (including, forexample, sucrose, glucose, dextrose and maltodextrin), polyethyleneglycol, waxes, natural and synthetic gums, polyvinylpyrrolidone,pregelatinized starch, povidone, cellulosic polymers (including, forexample, hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose(HPMC), methyl cellulose, hydroxyethyl cellulose, and the like),hydroxypropyl cellulose (HPC), and the like. Accordingly, in someembodiments, the formulations disclosed herein can include at least onebinder to enhance the compressibility of the major excipient(s). Forexample, the formulation can include at least one of the followingbinders in the following preferred ranges: from about 2% to about 6% w/whydroxypropyl cellulose (Klucel), from about 2% to about 5% w/wpolyvinylpyrrolidone (PVP), from about 1% to about 5% w/wmethycellulose, from about 2% to about 5% hydroxypropyl methycellulose,from about 1% to about 5% w/w ethylcellulose, from about 1% to about 5%w/w sodium carboxy methylcellulose, and the like. The above ranges areexemplary preferred ranges. One of ordinary skill in the art wouldrecognize additional binders and/or amounts that can be used in theformulations described herein. As would be recognized by one of ordinaryskill in the art, when incorporated into the formulations disclosedherein, the amounts of the major filler(s) and/or other excipients canbe reduced accordingly to accommodate the amount of binder added inorder to keep the overall unit weight of the tablet unchanged. In oneembodiment, the binder(s) is(are) sprayed on from solution, e.g. wetgranulation, to increase binding activity.

Disintegrants

In some embodiments, disintegrants are used, for example, to facilitatetablet disintegration after administration, and are generally starches,clays, celluloses, algins, gums, or crosslinked polymers. Suitabledisintegrants include, but are not limited to, crosslinkedpolyvinylpyrrolidone (PVP-XL), sodium starch glycolate, alginic acid,methacrylic acid DYB, microcrystalline cellulose, crospovidone,polacriline potassium, sodium starch glycolate, starch, pregelatinizedstarch, croscarmellose sodium, and the like. If desired, thepharmaceutical formulation can also contain minor amounts of nontoxicauxiliary substances such as wetting or emulsifying agents, pH bufferingagents and the like, for example, sodium acetate, sorbitan monolaurate,triethanolamine sodium acetate, triethanolamine oleate, sodium laurylsulfate, dioctyl sodium sulfosuccinate, polyoxyethylene sorbitan fattyacid esters, etc. and the like.

The above ranges are examples of preferred ranges. One of ordinary skillin the art would recognize additional disintegrants and/or amounts ofdisintegrants that can be used in the formulations described herein. Aswould be recognized by one of ordinary skill in the art, whenincorporated into the formulations disclosed herein, the amounts of themajor filler(s) and/or other excipients can be reduced accordingly toaccommodate the amount of disintegrant added in order to keep theoverall unit weight of the tablet unchanged.

Coatings

In some embodiments, the formulations can include a coating, forexample, a film coating. Where film coatings are involved, coatingpreparations can include, for example, a film-forming polymer, aplasticizer, or the like. Also, the coatings can include pigments and/oropacifiers. Non-limiting examples of film-forming polymers includehydroxypropyl methylcellulose, hydroxypropyl cellulose, methylcellulose,polyvinyl pyrrolidine, and starches. Non-limiting examples ofplasticizers include polyethylene glycol, tributyl citrate, dibutylsebecate, castor oil, and acetylated monoglyceride. Furthermore,non-limiting examples of pigments and opacifiers include iron oxides ofvarious colors, lake dyes of many colors, titanium dioxide, and thelike.

Diluents

In some embodiments, diluents are used, and are generally selected fromone or more of the compounds sucrose, fructose, glucose, galactose,lactose, maltose, invert sugar, calcium carbonate, lactose, starch,microcrystalline cellulose, lactose monohydrate, calcium hydrogenphosphate, anhydrous calcium hydrogen phosphate, a pharmaceuticallyacceptable polyol such as xylitol, sorbitol, maltitol, mannitol, isomaltand glycerol, polydextrose, starch, or the like, or any mixture thereof.

Surfactants

In some embodiments, surfactants are used. The use of surfactants aswetting agents in oral drug forms is described in the literature, forexample in H. Sucker, P. Fuchs, P. Speiser, Pharmazeutische Technologie,2nd edition, Thieme 1989, page 260. It is known from other papers, suchas published in Advanced Drug Delivery Reviews (1997), 23, pages163-183, that it is also possible to use surfactants, inter alia, toimprove the permeation and bioavailability of pharmaceutical activecompounds. Examples of surfactants include, but are not limited to,anionic surfactants, non-ionic surfactants, zwitterionic surfactants anda mixture thereof. Preferably, the surfactants is selected from thegroup consisting of poly(oxyethylene) sorbitan fatty acid ester,poly(oxyethylene) stearate, poly(oxyethylene) alkyl ether,polyglycolated glyceride, poly(oxyethylene) caster oil, sorbitan fattyacid ester, poloxamer, fatty acid salt, bile salt, alkyl sulfate,lecithin, mixed micelle of bile salt and lecithin, glucose ester vitaminE TPGS (D-α-tocopheryl polyethylene glycol 1000 succinate), sodiumlauryl sulfate, and the like, and a mixture thereof.

Glidants

In some embodiments, glidants are used. Examples of glidants which maybe used include, but are not limited to, colloidal silicon dioxide,magnesium trisilicate, powdered cellulose, starch, talc and calciumphosphate, or the like, and mixtures thereof.

The above excipients can be present in amount up to about 95% of thetotal composition weight, or up to about 85% of the total compositionweight, or up to about 75% of the total composition weight, or up toabout 65% of the total composition weight, or up to about 55% of thetotal composition weight, or up to about 45% of the total compositionweight, or up to about 43% of the total composition weight, or up toabout 40% of the total composition weight, or up to about 35% of thetotal composition weight, or up to about 30% of the total compositionweight, or up to about 25% of the total composition weight, or up toabout 20% of the total composition weight, or up to about 15% of thetotal composition weight, or up to about 10% of the total compositionweight, or less.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, topical, or intestinal administration; parenteraldelivery, including intramuscular, subcutaneous, intravenous,intramedullary injections, as well as intrathecal, directintraventricular, intraperitoneal, intranasal, or intraocularinjections. The compound of formula I can also be administered insustained or controlled release dosage forms, including depotinjections, osmotic pumps, pills, transdermal (includingelectrotransport) patches, and the like, for prolonged and/or timed,pulsed administration at a predetermined rate.

The pharmaceutical compositions of the present disclosure may bemanufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or tabletting processes.

Pharmaceutical compositions for use in accordance with the presentdisclosure thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen. Any of the well-knowntechniques, carriers, and excipients may be used as suitable and asunderstood in the art; e.g., in Remington's Pharmaceutical Sciences,above.

Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Suitableexcipients are, for example, water, saline, dextrose, mannitol, lactose,lecithin, albumin, sodium glutamate, cysteine hydrochloride, and thelike. In addition, if desired, the injectable pharmaceuticalcompositions may contain minor amounts of nontoxic auxiliary substances,such as wetting agents, pH buffering agents, and the like.Physiologically compatible buffers include, but are not limited to,Hanks's solution, Ringer's solution, or physiological saline buffer. Ifdesired, absorption enhancing preparations (for example, liposomes), maybe utilized.

For transmucosal administration, penetrants appropriate to the barrierto be permeated may be used in the formulation.

Pharmaceutical formulations for parenteral administration, e.g., bybolus injection or continuous infusion, include aqueous solutions of theactive compounds in water-soluble form. Additionally, suspensions of theactive compounds may be prepared as appropriate oily injectionsuspensions. Suitable lipophilic solvents or vehicles include fatty oilssuch as sesame oil, or other organic oils such as soybean, grapefruit oralmond oils, or synthetic fatty acid esters, such as ethyl oleate ortriglycerides, or liposomes. Aqueous injection suspensions may containsubstances which increase the viscosity of the suspension, such assodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, thesuspension may also contain suitable stabilizers or agents that increasethe solubility of the compounds to allow for the preparation of highlyconcentrated solutions. Formulations for injection may be presented inunit dosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

For oral administration, the compound of formula I can be formulatedreadily by combining the active compound with pharmaceuticallyacceptable carriers well known in the art. Such carriers enable thecompound of formula I to be formulated as tablets, film coated tablets,pills, dragees, capsules, liquids, gels, get caps, pellets, beads,syrups, slurries, suspensions and the like, for oral ingestion by apatient to be treated. Pharmaceutical preparations for oral use can beobtained by combining the active compound with solid excipient,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable auxiliaries, if desired, to obtaintablets or dragee cores. Suitable excipients are, in particular, fillerssuch as sugars, including lactose, sucrose, mannitol, or sorbitol;cellulose preparations such as, for example, maize starch, wheat starch,rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Dragee cores areprovided with suitable coatings. For this purpose, concentrated sugarsolutions may be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses. For this purpose, concentratedsugar solutions may be used, which may optionally contain gum arabic,talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses. In addition, stabilizers can beadded. All formulations for oral administration should be in dosagessuitable for such administration. In some embodiments, formulations ofthe compound of formula I with an acceptable immediate releasedissolution profile and a robust, scalable method of manufacture aredisclosed.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compound of formula I isconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

Further disclosed herein are various pharmaceutical compositions wellknown in the pharmaceutical art for uses that include intraocular,intranasal, and intraauricular delivery. Suitable penetrants for theseuses are generally known in the art. Pharmaceutical compositions forintraocular delivery include aqueous ophthalmic solutions of the activecompounds in water-soluble form, such as eyedrops, or in gellan gum(Shedden et al., Clin. Ther., 23(3):440-50 (2001)) or hydrogels (Mayeret al., Ophthalmologica, 210(2):101-3 (1996)); ophthalmic ointments;ophthalmic suspensions, such as microparticulates, drug-containing smallpolymeric particles that are suspended in a liquid carrier medium(Joshi, A., J. Ocul. Pharmacol., 10(1):29-45 (1994)), lipid-solubleformulations (Alm et al., Prog. Clin. Biol. Res., 312:447-58 (1989)),and microspheres (Mordenti, Toxicol. Sci., 52(1):101-6 (1999)); andocular inserts. All of the above-mentioned references are incorporatedherein by reference in their entireties. Such suitable pharmaceuticalformulations are most often and preferably formulated to be sterile,isotonic and buffered for stability and comfort. Pharmaceuticalcompositions for intranasal delviery may also include drops and spraysoften prepared to simulate in many respects nasal secretions to ensuremaintenance of normal ciliary action. As disclosed in Remington'sPharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa.(1990), which is incorporated herein by reference in its entirety, andwell-known to those skilled in the art, suitable formulations are mostoften and preferably isotonic, slightly buffered to maintain a pH of 5.5to 6.5, and most often and preferably include antimicrobialpreservatives and appropriate drug stabilizers. Pharmaceuticalformulations for intraauricular delivery include suspensions andointments for topical application in the ear. Common solvents for suchaural formulations include glycerin and water.

The compound of formula I may also be formulated in rectal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compound offormula I may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compound of formula I may be formulated with suitablepolymeric or hydrophobic materials (for example as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

For hydrophobic compounds, a suitable pharmaceutical carrier may be acosolvent system comprising benzyl alcohol, a nonpolar surfactant, awater-miscible organic polymer, and an aqueous phase. A common cosolventsystem used is the VPD co-solvent system, which is a solution of 3% w/vbenzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™, and65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.Naturally, the proportions of a co-solvent system may be variedconsiderably without destroying its solubility and toxicitycharacteristics. Furthermore, the identity of the co-solvent componentsmay be varied: for example, other low-toxicity nonpolar surfactants maybe used instead of POLYSORBATE 80™; the fraction size of polyethyleneglycol may be varied; other biocompatible polymers may replacepolyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars orpolysaccharides may substitute for dextrose.

Alternatively, other delivery systems for hydrophobic pharmaceuticalcompounds may be employed. Liposomes and emulsions are well knownexamples of delivery vehicles or carriers for hydrophobic drugs. Certainorganic solvents such as dimethylsulfoxide also may be employed,although usually at the cost of greater toxicity. Additionally, thecompounds may be delivered using a sustained-release system, such assemipermeable matrices of solid hydrophobic polymers containing thetherapeutic agent. Various sustained-release materials have beenestablished and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the therapeuticreagent, additional strategies for protein stabilization may beemployed.

Agents intended to be administered intracellularly may be administeredusing techniques well known to those of ordinary skill in the art. Forexample, such agents may be encapsulated into liposomes. All moleculespresent in an aqueous solution at the time of liposome formation areincorporated into the aqueous interior. The liposomal contents are bothprotected from the external micro-environment and, because liposomesfuse with cell membranes, are efficiently delivered into the cellcytoplasm. The liposome may be coated with a tissue-specific antibody.The liposomes will be targeted to and taken up selectively by thedesired organ. Alternatively, small hydrophobic organic molecules may bedirectly administered intracellularly.

Additional therapeutic or diagnostic agents may be incorporated into thepharmaceutical compositions. Alternatively or additionally,pharmaceutical compositions may be combined with other compositions thatcontain other therapeutic or diagnostic agents.

Methods of Administration

The compound of formula I or pharmaceutical compositions may beadministered to the patient by any suitable means. Non-limiting examplesof methods of administration include, among others, (a) administrationthough oral pathways, which includes administration in capsule, tablet,granule, spray, syrup, or other such forms; (b) administration throughnon-oral pathways such as rectal, vaginal, intraurethral, intraocular,intranasal, or intraauricular, which includes administration as anaqueous suspension, an oily preparation or the like or as a drip, spray,suppository, salve, ointment or the like; (c) administration viainjection, subcutaneously, intraperitoneally, intravenously,intramuscularly, intradermally, intraorbitally, intracapsularly,intraspinally, intrasternally, or the like, including infusion pumpdelivery; (d) administration locally such as by injection directly inthe renal or cardiac area, e.g., by depot implantation; as well as (e)administration topically; as deemed appropriate by those of skill in theart for bringing the compound of formula I into contact with livingtissue.

Pharmaceutical compositions suitable for administration includecompositions where the compound of formula I is contained in an amounteffective to achieve its intended purpose. The therapeutically effectiveamount of the compound of formula I disclosed herein required as a dosewill depend on the route of administration, the type of animal,including human, being treated, and the physical characteristics of thespecific animal under consideration. The dose can be tailored to achievea desired effect, but will depend on such factors as weight, diet,concurrent medication and other factors which those skilled in themedical arts will recognize. More specifically, a therapeuticallyeffective amount means an amount of compound effective to prevent,alleviate or ameliorate symptoms of disease or prolong the survival ofthe subject being treated. Determination of a therapeutically effectiveamount is well within the capability of those skilled in the art,especially in light of the detailed disclosure provided herein.

As will be readily apparent to one skilled in the art, the useful invivo dosage to be administered and the particular mode of administrationwill vary depending upon the age, weight and mammalian species treated,and the specific use for which the compound of formula I are employed.The determination of effective dosage levels, that is the dosage levelsnecessary to achieve the desired result, can be accomplished by oneskilled in the art using routine pharmacological methods. Typically,human clinical applications of products are commenced at lower dosagelevels, with dosage level being increased until the desired effect isachieved. Alternatively, acceptable in vitro studies can be used toestablish useful doses and routes of administration of the compositionsidentified by the present methods using established pharmacologicalmethods.

In non-human animal studies, applications of potential products arecommenced at higher dosage levels, with dosage being decreased until thedesired effect is no longer achieved or adverse side effects disappear.The dosage may range broadly, depending upon the desired effects and thetherapeutic indication. Typically, dosages may be between about 10microgram/kg and 100 mg/kg body weight, preferably between about 100microgram/kg and 10 mg/kg body weight. Alternatively dosages may bebased and calculated upon the surface area of the patient, as understoodby those of skill in the art.

Depending on the severity and responsiveness of the condition to betreated, dosing can also be a single administration of a slow releasecomposition, with course of treatment lasting from several days toseveral weeks or until cure is effected or diminution of the diseasestate is achieved. The amount of a composition to be administered will,of course, be dependent on many factors including the subject beingtreated, the severity of the affliction, the manner of administration,the judgment of the prescribing physician. The compound of formula I maybe administered orally or via injection at a dose from 0.001 to 2500mg/kg of the patient's body weight per day. The dose range for adulthumans is generally from 0.01 mg to 10 g/day. Tablets or other forms ofpresentation provided in discrete units may conveniently contain anamount of the compound of formula I which is effective at such dosage oras a multiple of the same, for instance, units containing 5 mg to 1000mg, usually from about 100 mg to about 800 mg. The precise amount ofcompound administered to a patient will be the responsibility of theattendant physician. However, the dose employed will depend on a numberof factors, including the age and sex of the patient, the precisedisorder being treated, and its severity. Also, the route ofadministration may vary depending on the condition and its severity.

The exact formulation, route of administration and dosage for thepharmaceutical compositions of the compound of formula I can be chosenby the individual physician in view of the patient's condition. (See,e.g., Fingl et al. 1975, in “The Pharmacological Basis of Therapeutics,”which is hereby incorporated herein by reference, with particularreference to Ch. 1). Typically, the dose range of the compositionadministered to the patient can be from about 0.01 to about 1000 mg/kgof the patient's body weight. The dosage may be a single one or a seriesof two or more given in the course of one or more days, as is needed bythe patient. In instances where human dosages for compounds have beenestablished for at least some condition, the present disclosure will usethose same dosages, or dosages that are between about 0.1% and about500%, more preferably between about 25% and about 250% of theestablished human dosage. Where no human dosage is established, as willbe the case for newly-discovered pharmaceutical compounds, a suitablehuman dosage can be inferred from ED₅₀ or ID₅₀ values, or otherappropriate values derived from in vitro or in vivo studies, asqualified by toxicity studies and efficacy studies in animals.

It should be noted that the attending physician would know how to andwhen to terminate, interrupt, or adjust administration due to toxicityor organ dysfunctions. Conversely, the attending physician would alsoknow to adjust treatment to higher levels if the clinical response werenot adequate (precluding toxicity). The magnitude of an administrateddose in the management of the disorder of interest will vary with theseverity of the condition to be treated and to the route ofadministration. The severity of the condition may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and perhaps dose frequency, will also vary according to theage, body weight, and response of the individual patient. A programcomparable to that discussed above may be used in veterinary medicine.

Although the exact dosage will be determined on a drug-by-drug basis, inmost cases, some generalizations regarding the dosage can be made. Thedaily dosage regimen for an adult human patient may be, for example, anoral dose of between 0.1 mg and 2000 mg of each active ingredient,preferably between 1 mg and 1500 mg, e.g., 5 to 1000 mg. In otherembodiments, an oral dose of each active ingredient of between 1 mg and1000 mg, preferably between 50 mg and 900 mg, e.g., 100 to 800 mg isused. In cases of administration of a pharmaceutically acceptable salt,dosages may be calculated as the free base. In some embodiments, thecomposition is administered 1 to 4 times per day. Alternatively thecompositions of the compound of formula I may be administered bycontinuous intravenous infusion, preferably at a dose of each activeingredient up to 1000 mg per day. As will be understood by those ofskill in the art, in certain situations it may be necessary toadminister the compound disclosed herein in amounts that exceed, or evenfar exceed, the above-stated, preferred dosage range in order toeffectively and aggressively treat particularly aggressive diseases orinfections. In some embodiments, the compound of formula I will beadministered for a period of continuous therapy, for example for a weekor more, or for months or years.

In some embodiments, the dosing regimen of the compound of formula I isadministered for a period of time, which time period can be, forexample, from at least about 4 weeks to at least about 8 weeks, from atleast about 4 weeks to at least about 12 weeks, from at least about 4weeks to at least about 16 weeks, or longer. The dosing regimen of thecompound of formula I can be administered three times a day, twice aday, daily, every other day, three times a week, every other week, threetimes per month, once monthly, substantially continuously orcontinuously.

Some embodiments provide a method to use an effective amount of thecompound of formula I in the treatment of endothelin-dependent orangiotensin II-dependent disorders in a patient comprising administeringto the patient a dosage of the compound of formula I containing anamount of about 10 mg to about 1000 mg, of drug per dose of the compoundof formula I, orally, three times per month, once monthly, once weekly,once every three days, once every two days, once per day, twice per day,or three times per day substantially continuously or continuously, forthe desired duration of treatment.

Some embodiments provide a method to use an effective amount of thecompound of formula I in the treatment of endothelin-dependent orangiotensin II-dependent disorders in a patient comprising administeringto the patient a dosage of the compound of formula I containing anamount of about 100 mg to about 1000 mg, of drug per dose of thecompound of formula I, orally, three times per month, once monthly, onceweekly, once every three days, once every two days, once per day, twiceper day, or three times per day substantially continuously orcontinuously, for the desired duration of treatment.

Some embodiments provide a method to use an effective amount of thecompound of formula I in the treatment of endothelin-dependent orangiotensin II-dependent disorders in a patient comprising administeringto the patient a dosage of the compound of formula I containing anamount of about 100 mg to about 900 mg, of drug per dose of the compoundof formula I, orally, three times per month, once monthly, once weekly,once every three days, once every two days, once per day, twice per day,or three times per day substantially continuously or continuously, forthe desired duration of treatment.

Some embodiments provide a method to use an effective amount of thecompound of formula I in the treatment of endothelin-dependent orangiotensin II-dependent disorders in a patient comprising administeringto the patient a dosage of the compound of formula I containing anamount of about 100 mg, of drug per dose of the compound of formula I,orally, three times per month, once monthly, once weekly, once everythree days, once every two days, once per day, twice per day, or threetimes per day substantially continuously or continuously, for thedesired duration of treatment.

Some embodiments provide a method to use an effective amount of thecompound of formula I in the treatment of endothelin-dependent orangiotensin II-dependent disorders in a patient comprising administeringto the patient a dosage of the compound of formula I containing anamount of about 200 mg, of drug per dose of the compound of formula I,orally, three times per month, once monthly, once weekly, once everythree days, once every two days, once per day, twice per day, or threetimes per day substantially continuously or continuously, for thedesired duration of treatment.

Some embodiments provide a method to use an effective amount of thecompound of formula I in the treatment of endothelin-dependent orangiotensin II-dependent disorders in a patient comprising administeringto the patient a dosage of the compound of formula I containing anamount of about 400 mg, of drug per dose of the compound of formula I,orally, three times per month, once monthly, once weekly, once everythree days, once every two days, once per day, twice per day, or threetimes per day substantially continuously or continuously, for thedesired duration of treatment.

Some embodiments provide a method to use an effective amount of thecompound of formula I in the treatment of endothelin-dependent orangiotensin II-dependent disorders in a patient comprising administeringto the patient a dosage of the compound of formula I containing anamount of about 800 mg, of drug per dose of the compound of formula I,orally, three times per month, once monthly, once weekly, once everythree days, once every two days, once per day, twice per day, or threetimes per day substantially continuously or continuously, for thedesired duration of treatment.

Some embodiments provide a method to use an effective amount of thecompound of formula I in the treatment of endothelin-dependent orangiotensin II-dependent disorders in a patient comprising administeringto the patient a dosage of the compound of formula I containing anamount of about 0.01 mg to about 1000 mg of drug per kilogram of bodyweight per dose of the compound of formula I, orally, three times permonth, once monthly, once weekly, once every three days, once every twodays, once per day, twice per day, or three times per day substantiallycontinuously or continuously, for the desired duration of treatment.

Some embodiments provide a method to use an effective amount of thecompound of formula I in the treatment of endothelin-dependent orangiotensin II-dependent disorders in a patient comprising administeringto the patient a dosage of the compound of formula I containing anamount of about 0.1 mg to about 500 mg of drug per kilogram of bodyweight per dose of the compound of formula I, orally, three times permonth, once monthly, once weekly, once every three days, once every twodays, once per day, twice per day, or three times per day substantiallycontinuously or continuously, for the desired duration of treatment.

Some embodiments provide a method to use an effective amount of thecompound of formula I in the treatment of endothelin-dependent orangiotensin II-dependent disorders in a patient comprising administeringto the patient a dosage of the compound of formula I containing anamount of about 0.1 mg to about 100 mg of drug per kilogram of bodyweight per dose of the compound of formula I, orally, three times permonth, once monthly, once weekly, once every three days, once every twodays, once per day, twice per day, or three times per day substantiallycontinuously or continuously, for the desired duration of treatment.

Some embodiments provide a method to use an effective amount of thecompound of formula I in the treatment of endothelin-dependent orangiotensin II-dependent disorders in a patient comprising administeringto the patient a dosage of the compound of formula I containing anamount of about 0.5 mg of drug per kilogram of body weight per dose ofthe compound of formula I, orally, three times per month, once monthly,once weekly, once every three days, once every two days, once per day,twice per day, or three times per day substantially continuously orcontinuously, for the desired duration of treatment.

Some embodiments provide a method to use an effective amount of thecompound of formula I in the treatment of endothelin-dependent orangiotensin II-dependent disorders in a patient comprising administeringto the patient a dosage of the compound of formula I containing anamount of about 1 mg of drug per kilogram of body weight per dose of thecompound of formula I, orally, three times per month, once monthly, onceweekly, once every three days, once every two days, once per day, twiceper day, or three times per day substantially continuously orcontinuously, for the desired duration of treatment.

Some embodiments provide a method to use an effective amount of thecompound of formula I in the treatment of endothelin-dependent orangiotensin II-dependent disorders in a patient comprising administeringto the patient a dosage of the compound of formula I containing anamount of about 5 mg of drug per kilogram of body weight per dose of thecompound of formula I, orally, three times per month, once monthly, onceweekly, once every three days, once every two days, once per day, twiceper day, or three times per day substantially continuously orcontinuously, for the desired duration of treatment.

Some embodiments provide a method to use an effective amount of thecompound of formula I in the treatment of endothelin-dependent orangiotensin II-dependent disorders in a patient comprising administeringto the patient a dosage of the compound of formula I containing anamount of about 25 mg of drug per kilogram of body weight per dose ofthe compound of formula I, orally, three times per month, once monthly,once weekly, once every three days, once every two days, once per day,twice per day, or three times per day substantially continuously orcontinuously, for the desired duration of treatment.

Some embodiments provide a method to use an effective amount of thecompound of formula I in the treatment of endothelin-dependent orangiotensin dependent disorders in a patient comprising administering tothe patient a dosage of the compound of formula I containing an amountof about 50 mg of drug per kilogram of body weight per dose of thecompound of formula I, orally, three times per month, once monthly, onceweekly, once every three days, once every two days, once per day, twiceper day, or three times per day substantially continuously orcontinuously, for the desired duration of treatment.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain themodulating effects, or minimal effective concentration (MEC). The MECwill vary for each compound but can be estimated from in vitro data.Dosages necessary to achieve the MEC will depend on individualcharacteristics and route of administration. However, HPLC assays orbioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using MEC value. In someembodiments, compositions can be administered using a regimen whichmaintains plasma levels above the MEC for 10-90% of the time, forexample, between 15-30%, 20-45%, 25-50%, 30-55%, 35-60%, 40-65%, 45-70%,50-75%, 55-80%, 60-90%, 65-75%, 70-80%, 75-85%, 15-90%, 20-90%, 25-90%,30-90%, 35-90%, 40-90%, 45-90%, 50-90%, 55-90%, 60-90%, 65-90%, 70-90%,75-90%, or 80-90%. In some embodiments, compositions can be administeredusing a regimen which maintains plasma levels above the MEC for 20-90%of the time. In some embodiments, compositions can be administered usinga regimen which maintains plasma levels above the MEC for 30-90% of thetime, between 40-90% and most typically between 50-90%.

In cases of local administration or selective uptake, the effectivelocal concentration of the drug may not be related to plasmaconcentration.

The amount of composition administered may be dependent on the subjectbeing treated, on the subject's weight, the severity of the affliction,the manner of administration and the judgment of the prescribingphysician.

The compound of formula I disclosed herein can be evaluated for efficacyand toxicity using known methods. For example, the toxicology of thecompound of formula I may be established by determining in vitrotoxicity towards a cell line, such as a mammalian, and preferably human,cell line. The results of such studies are often predictive of toxicityin animals, such as mammals, or more specifically, humans.Alternatively, the toxicity of the compound of formula I in an animalmodel, such as mice, rats, rabbits, or monkeys, may be determined usingknown methods. The efficacy of the compound of formula I may beestablished using several recognized methods, such as in vitro methods,animal models, or human clinical trials. Recognized in vitro modelsexist for nearly every class of condition, including but not limited tocancer, cardiovascular disease, and various immune dysfunction.Similarly, acceptable animal models may be used to establish efficacy ofchemicals to treat such conditions. When selecting a model to determineefficacy, the skilled artisan can be guided by the state of the art tochoose an appropriate model, dose, and route of administration, andregime. Of course, human clinical trials can also be used to determinethe efficacy of a compound in humans.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration. The pack or dispensermay also be accompanied with a notice associated with the container inform prescribed by a governmental agency regulating the manufacture,use, or sale of pharmaceuticals, which notice is reflective of approvalby the agency of the form of the drug for human or veterinaryadministration. Such notice, for example, may be the labeling approvedby the U.S. Food and Drug Administration for prescription drugs, or theapproved product insert. Compositions comprising the compound of formulaI formulated in a compatible pharmaceutical carrier may also beprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

An effective amount of the compound of formula I may be determined byone of ordinary skill in the art, and includes exemplary dosage amountsfor a human of from about 0.1 to about 100 mg/kg, preferably about 0.2to about 50 mg/kg and more preferably from about 0.5 to about 25 mg/kgof body weight (or from about 1 to about 2500 mg, preferably from about100 to about 800 mg) of active compound per day, which may beadministered in a single dose or in the form of individual divideddoses, such as from 1 to 4 times per day. It will be understood that thespecific dose level and frequency of dosage for any particular subjectmay be varied and will depend upon a variety of factors including theactivity of the specific compound employed, the metabolic stability andlength of action of that compound, the species, age, body weight,general health, sex and diet of the subject, the mode and time ofadministration, rate of excretion, drug combination, and severity of theparticular condition. Preferred subjects for treatment include animals,most preferably mammalian species such as humans, and domestic animalssuch as dogs, cats and the like, subject to endothelin-dependent orangiotensin II dependent disorders.

Pharmaceutical compositions comprising the compound of formula I capableof treating an endothelin-dependent or angiotensin II-dependent disorderin an amount effective therefore, and a pharmaceutically acceptablevehicle or diluent are also disclosed. The compositions of the presentdisclosure may contain other therapeutic agents as described below, andmay be formulated, for example, by employing conventional solid orliquid vehicles or diluents, as well as pharmaceutical additives of atype appropriate to the mode of desired administration (for example,excipients, binders, preservatives, stabilizers, flavors, etc.)according to techniques such as those well known in the art ofpharmaceutical formulation or called for by accepted pharmaceuticalpractice.

The compound of formula I may be administered by any suitable means, forexample, orally, such as in the form of tablets, capsules, granules orpowders; sublingually; buccally; parenterally, such as by subcutaneous,intravenous, intramuscular, or intrasternal injection or infusiontechniques (e.g., as sterile injectable aqueous or non-aqueous solutionsor suspensions); nasally such as by inhalation spray; topically, such asin the form of a cream or ointment; or rectally such as in the form ofsuppositories; in dosage unit formulations containing non-toxic,pharmaceutically acceptable vehicles or diluents. In some embodiments,the amount of the compound of formula I administered in the formulationcan be 800 mg per unit dosage. In some embodiments, the amount of thecompound of formula I administered per day can be about 800 mg. Forexample, about 400 mg of the compound can be administered in aformulation twice a day, about 200 mg of the compound in the formulationcan be administered in a formulation four times a day, about 100 mg ofthe compound in the formulation can be administered in a formulationeight times a day.

The compound of formula I, for example, be administered in a formsuitable for immediate release or extended release. Immediate release orextended release may be achieved by the use of suitable pharmaceuticalcompositions comprising the compound of formula I, or, particularly inthe case of extended release, by the use of devices such as subcutaneousimplants or osmotic pumps. In some embodiments, the amount of thecompound of formula I, in the immediate release formulation, can beabout 800 mg per unit dosage. In some embodiments, the amount of thecompound of formula I, in the immediate release formulation,administered per day can be about 800 mg. For example, about 400 mg ofthe compound can be administered in an immediate release formulationtwice a day, about 200 mg of the compound in an immediate releaseformulation can be administered four times a day, about 100 mg of thecompound in an immediate release formulation can be administered eighttimes a day.

The compound of formula I may also be administered liposomally. Forexample, the active substance can be utilized in a composition such astablet, capsule, solution or suspension containing about 5 mg to about1000 mg per unit dosage of the compound of formula I or in topical formfor wound healing (0.01 to 5% by weight the compound of formula I, 1 to5 treatments per day). In some embodiments, the amount of the compoundof formula I, administered liposomally, can be about 800 mg per unitdosage. In some embodiments, the amount of the compound of formula I, inthe liposome formulation, administered per day can be about 800 mg. Forexample, about 400 mg of the compound can be administered in a liposomeformulation twice a day, about 200 mg of the compound in a liposomeformulation can be administered four times a day, about 100 mg of thecompound in a liposome formulation can be administered eight times aday.

The compound of formula I may be compounded in a conventional mannerwith a physiologically acceptable vehicle or carrier, excipient, binder,preservative, stabilizer, flavor, etc., or with a topical carrier. Insome embodiments, the amount of the compound of formula I can be about800 mg per unit dosage. In some embodiments, the amount of the compoundof formula I administered per day can be about 800 mg. For example,about 400 mg of the compound can be administered twice a day, about 200mg of the compound can be administered four times a day, about 100 mg ofthe compound can be administered eight times a day.

The compound of formula I can also be formulated in compositions such assterile solutions or suspensions for parenteral administration. About0.1 to about 800 milligrams of the compound of formula I, typically 200milligrams, more typically about 400 milligrams, most typically about800 milligrams, may be compounded with a physiologically acceptablevehicle, carrier, excipient, binder, preservative, stabilizer, etc., ina unit dosage form as called for by accepted pharmaceutical practice.The amount of active substance in these compositions or preparations ispreferably such that a suitable dosage in the range indicated isobtained. In some embodiments, the amount of the compound of formula I,administrated parenterally, can be about 800 mg per unit dosage. In someembodiments, the amount of compound of formula I administered per daycan be about 800 mg. For example, about 400 mg of the compound can beadministered twice a day, about 200 mg of the compound can beadministered four times a day, or about 100 mg of the compound can beadministered eight times a day.

Exemplary compositions for oral administration include suspensions whichmay contain, for example, microcrystalline cellulose for imparting bulk,alginic acid or sodium alginate as a suspending agent, methylcelluloseas a viscosity enhancer, and sweeteners or flavoring agents such asthose known in the art; and immediate release tablets which may contain,for example, microcrystalline cellulose, dicalcium phosphate, starch,magnesium stearate and/or lactose and/or other excipients, binders,extenders, disintegrants, diluents and lubricants such as those known inthe art. Molded tablets, compressed tablets or freeze-dried tablets areexemplary forms which may be used. Exemplary compositions include thoseformulating the compound of formula I with fast dissolving diluents suchas mannitol, lactose, sucrose and/or cyclodextrins. Also included insuch formulations may be high molecular weight excipients such ascelluloses (avicel) or polyethylene glycols (PEG). Such formulations mayalso include an excipient to aid mucosal adhesion such as hydroxy propylcellulose (HPC), hydroxy propyl methyl cellulose (HPMC), sodium carboxymethyl cellulose (SCMC), maleic anhydride copolymer (e.g., Gantrez), andagents to control release such as polyacrylic copolymer (e.g., Carbopol934). Lubricants, glidants, flavors, coloring agents and stabilizers mayalso be added for ease of fabrication and use.

Exemplary compositions for nasal aerosol or inhalation administrationinclude solutions in saline which may contain, for example, benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, and/or other solubilizing or dispersing agents such asthose known in the art.

Exemplary compositions for parenteral administration include injectablesolutions or suspensions which may contain, for example, suitablenon-toxic, parenterally acceptable diluents or solvents, such asmannitol, 1,3-butanediol, water, Ringer's solution, an isotonic sodiumchloride solution, or other suitable dispersing or wetting andsuspending agents, including synthetic mono- or diglycerides, and fattyacids, including oleic acid.

Exemplary compositions for rectal administration include suppositorieswhich may contain, for example, a suitable non-irritating excipient,such as cocoa butter, synthetic glyceride esters or polyethyleneglycols, which are solid at ordinary temperatures, but liquify and/ordissolve in the rectal cavity to release the drug.

Exemplary compositions for topical administration include a topicalcarrier such as Plastibase (mineral oil gelled with polyethylene). Forexample, the compound of formula I may be administered topically totreat peripheral vascular diseases and as such may be formulated as acream or ointment.

The compound of formula I may be employed alone or in combination withother suitable therapeutic agents useful in the treatment ofendothelin-dependent or angiotensin II-dependent disorders. For example,the compound of formula I can be formulated in combination withendothelin converting enzyme (ECE) inhibitors, such as phosphoramidon;thromboxane receptor antagonists such as ifetroban; potassium channelopeners; thrombin inhibitors (e.g., hirudin and the like); growth factorinhibitors such as modulators of PDGF activity; platelet activatingfactor (PAF) antagonists; anti-platelet agents such as GPIIb/IIIablockers (e.g., abciximab, eptifibatide, and tirofiban), P2Y(AC)antagonists (e.g., clopidogrel, ticlopidine and CS-747), and aspirin;anticoagulants such as warfarin, low molecular weight heparins such asenoxaparin, Factor Vila inhibitors, and Factor Xa inhibitors such asthose described in U.S. Pat. No. 6,297,233, issued Oct. 2, 2001; renininhibitors; angiotensin converting enzyme (ACE) inhibitors such ascaptopril, zofenopril, fosinopril, ceranapril, alacepril, enalapril,delapril, pentopril, quinapril, ramipril, lisinopril and salts of suchcompounds; neutral endopeptidase (NEP) inhibitors; vasopepsidaseinhibitors (dual NEP-ACE inhibitors) such as omapatrilat andgemopatrilat; HMG CoA reductase inhibitors such as pravastatin,lovastatin, atorvastatin, simvastatin, NK-104 (a.k.a. itavastatin, ornisvastatin or nisbastatin) and ZD-4522 (a.k.a. rosuvastatin, oratavastatin or visastatin); squalene synthetase inhibitors; fibrates;bile acid sequestrants such as questran; niacin; anti-atheroscleroticagents such as ACAT inhibitors; MTP inhibitors such as those describedin U.S. Ser. No. 09/007,938 filed Jan. 16, 1998; calcium channelblockers such as amlodipine besylate; potassium channel activators;alpha-adrenergic agents, beta-adrenergic agents such as carvedilol andmetoprolol; antiarrhythmic agents; diuretics, such as chlorothiazide,hydrochlorothiazide, flumethiazide, hydroflumethiazide,bendroflumethiazide, methylchlorothiazide, trichloromethiazide,polythiazide or benzothiazide as well as ethacrynic acid, tricrynafen,chlorthalidone, furosemide, musolimine, bumetanide, triamterene,amiloride and spironolactone and salts of such compounds; thrombolyticagents such as tissue plasminogen activator (tPA), recombinant tPA,streptokinase, urokinase, prourokinase and anisoylated plasminogenstreptokinase activator complex (APSAC); anti-diabetic agents such asbiguanides (e.g. metformin), glucosidase inhibitors (e.g., acarbose),insulins, meglitinides (e.g., repaglinide), sulfonylureas (e.g.,glimepiride, glyburide, and glipizide), biguanide/glyburide combinationssuch as those described in U.S. Pat. No. 6,586,438, issued Jul. 1, 2003and U.S. Pat. No. 7,598,262, issued Oct. 6, 2009; thiozolidinediones(e.g. troglitazone, rosiglitazone and pioglitazone), and PPAR-gammaagonists; mineralocorticoid receptor antagonists such as spironolactoneand eplerenone; growth hormone secretagogues such as those described inU.S. Pat. No. 6,380,184 issued Apr. 30, 2002 and U.S. Pat. No. 6,518,292filed Feb. 11, 2003; aP2 inhibitors such as those described in U.S. Pat.No. 7,390,824 filed Jun. 24, 2008 and U.S. Ser. No. 09/390,275 filedSep. 7, 1999; digitalis; ouabian; non-steroidal antiinflammatory drugs(NSAIDS) such as aspirin and ibuprofen; phosphodiesterase inhibitorssuch as PDE III inhibitors (e.g., cilostazol) and PDE V inhibitors(e.g., sildenafil); protein tyrosine kinase inhibitors;antiinflammatories; antiproliferatives such as methotrexate, FK506(tacrolimus, Prograf), mycophenolate and mofetil; chemotherapeuticagents; immunosuppressants; anticancer agents and cytotoxic agents(e.g., alkylating agents, such as nitrogen mustards, alkyl sulfonates,nitrosoureas, ethylenimines, and triazenes); antimetabolites such asfolate antagonists, purine analogues, and pyrimidine analogues;antibiotics, such as anthracyclines, bleomycins, mitomycin,dactinomycin, and plicamycin; enzymes, such as L-asparaginase;farnesyl-protein transferase inhibitors; hormonal agents, such asglucocorticoids (e.g., cortisone), estrogens/antiestrogens,androgens/antiandrogens, progestins, and luteinizing hormone-releasinghormone anatagonists, octreotide acetate; microtubule-disruptor agents,such as ecteinascidins or their analogs and derivatives;microtubule-stabilizing agents such as paclitaxel (Taxol®), docetaxel(Taxotere®), and epothilones A-F or their analogs or derivatives;plant-derived products, such as vinca alkaloids, epipodophyllotoxins,taxanes; and topoisomerase inhibitors; prenyl-protein transferaseinhibitors; and miscellaneous agents such as, hydroxyurea, procarbazine,mitotane, hexamethylmelamine, platinum coordination complexes such ascisplatin and carboplatin); cyclosporins; steroids such as prednisone ordexamethasone; gold compounds; cytotoxic drugs such as azathiprine andcyclophosphamide; TNF-alpha inhibitors such as tenidap; anti-TNFantibodies or soluble TNF receptor such as etanercept (Enbrel) rapamycin(sirolimus or Rapamune), leflunimide (Arava); and cyclooxygenase-2(COX-2) inhibitors such as celecoxib (Celebrex) and rofecoxib (Vioxx).

If formulated as a fixed dose, such combination products employ thecompound of formula I within the dosage range described below and theother pharmaceutically active agent within its approved dosage range.The compound of formula I may also be formulated with, or useful inconjunction with, antifungal and immunosuppressive agents such asamphotericin B, cyclosporins and the like to counteract the glomerularcontraction and nephrotoxicity secondary to such compounds. The compoundof formula I may also be used in conjunction with hemodialysis.

The above other therapeutic agents, when employed in combination withthe compound of formula I, may be used, for example, in those amountsindicated in the Physicians' Desk Reference (PDR) or as otherwisedetermined by one of ordinary skill in the art.

The following assays may be employed in ascertaining the degree ofactivity of a compound (“drug”) as an endothelin and angiotensin IIreceptor antagonist. The compound of formula I described in thefollowing Examples has been tested in these assays, and has shownactivity.

ET_(A/B) Attached Cell Binding Assay

CHO-K1 cells expressing either the human endothelin A or endothelin Breceptor were cultured in Ham's F12 media (Gibco/BRL, Grand Island,N.Y.) with 10% fetal bovine serum (Hyclone), supplemented with 300 μg/mLGeneticin (G-418 Gibco BRL Products, Grand Island, N.Y.) and maintainedat 37° C. with 5% CO₂ in a humidified incubator. Twenty four hours priorto assay, the cells were treated with 0.25% trypsin-EDTA and were seededin Falcon, 96 well tissue culture plates at a density of 1.8×10⁴cells/well (the monolayer should reach 80-90% confluency by the day ofassay).

In the attached cell assay, culture media was aspirated from each welland the monolayers were washed with 50 μL of PBS (Mg⁺⁺, Ca⁺⁺ free). Thebinding assay was performed in a total volume of 125 μL consisting ofassay buffer (50 mM Tris, pH 7.4, including 1% BSA, and 2 μMphosphoramidon), and 25 μL of either 500 nM ET-1 (to define nonspecificbinding) or competing drug. The reaction was initiated with the additionof 25 μL of 0.25 nM [¹²⁵I]-ET-1 (New England Nuclear). Incubation wascarried out with gentle orbital shaking, at 4° C., reaching equilibriumat 4 hours. The reaction was terminated by aspiration of the reactionbuffer and two subsequent washes with cold PBS (Mg⁺⁺, Ca⁺⁺ free). Thecells were dissociated by the addition of 100 μL of 0.5N NaOH followedby incubation for 40 minutes. Samples were then transferred from the 96well format into tubes for counting in a Cobra gamma counter (Packard).Data was analyzed with curve fitting software by Sigma plot.

RASMC Binding Assay

Assays were conducted in a total volume of 250 μL in 96 well microtitreplates. The incubation mixture contained 50 μL[125]I-Sar-Ile-Angiotensin II (0.2 nM), 25 μL of drug dissolved in DMSO,or angiotensin II (1 μM) to define non-specific binding. Binding to rataortic smooth muscle cells (RASMCs) was conducted in RPMI media (GibcoBRL Products, Grand Island, N.Y.) containing 0.1% BSA for 2 hours atroom temperature with continuous shaking. Unbound radioligand was washedfrom the wells. The RASMCs with bound radioligand are lysed with 1%Triton X and 0.1% BSA in distilled water for 15 minutes at roomtemperature with continuous shaking. The solution in each well wastransferred to tubes and placed in a gamma counter.

All documents cited in the present specification are incorporated hereinby reference in their entirety.

The following Examples illustrate embodiments of the present disclosure,and are not intended to limit the scope of the claims. Abbreviationsemployed herein are defined below.

Abbreviations

Ac=acetyl

BOC=tert-butoxycarbonyl

n-Bu=n-butyl

BSA=bovine serum albumin

bp=boiling point

CDI=1,1′ carbonyldiimidazole

d=days

DIBAL-H=diisobutylaluminum hydride

DMF=N,N-dimethylformamide

DMSO=dimethylsulfoxide

EDTA=ethylenediaminetetraacetic acid

eq=equivalents

Et=ethyl

ET=endothelin

ET-1=endothelin-1

EtOAc=ethyl acetate

EtOH=ethanol

g=grams

h=hours

kg=kilograms

Me=methyl

MEM=methoxyethoxymethyl

MeOH=methanol

m2/g=square meters per gram, is used as a measurement of particlesurface area

min=minutes

mL=milliliters

mmol=millimoles

mm Hg=millimeters of mercury

RH=relative humidity

MOM=methoxymethyl

mp=melting point

Ms=methanesulfonyl

NBS=N-bromosuccinimide

° C.=degrees Celsius

° F.=degrees Fahrenheit

PBS=phosphate buffered saline

Ph=phenyl

n-Pr=n-propyl

μL=microliters

μg=micrograms

SEM=2-(trimethylsiloxy)ethoxymethyl

rt=room temperature

TFA=trifluoroacetic acid

THF=tetrahydrofuran

General Methods

The following General Methods were employed in the Preparations andExamples.

General Method 1: Alkylation of Heterocycles or Aliphatic Alcohols

RCH₂X→RCH₂—OCH₂CH₃ or RCH₂-heterocycle

X=Br or MsO

Sodium hydride (60% dispersion in mineral oil, 1.2 eq) was added at 0°C. to a 1.0 M solution or suspension of an appropriate heterocycle orethyl alcohol (1.5 eq) in DMF. The mixture was allowed to warm to rt,was stirred for 20 min, and was then cooled back to 0° C. To theheterocycle mixture was added a solution of the appropriate alkylbromide or alkyl methanesulfonate (1.0 eq) in a minimal amount of DMF.The resultant mixture was allowed to warm to rt and was stirred for16-24 h. The reaction mixture was diluted with EtOAc and washed withwater and brine. The organic layer was dried over sodium sulfate andconcentrated, and the residue chromatographed on silica gel withhexanes/ethyl acetate as eluant to yield the alkylation product.

General Method 2: Reduction of an Aromatic Nitrile to an AromaticAldehyde Using DIBAL-H

ArCN→ArCHO

DIBAL-H (1.5 M solution in toluene, 1.5 eq) was added dropwise at 0° C.to a 0.5 M solution of an aromatic nitrile (1.0 eq) in toluene or 9:1toluene/dichloromethane. The solution was stirred at 0° C. for 1-4 h,and was then treated with excess methanol. After 15 min, 2N hydrochloricacid was added and the mixture was stirred vigorously for an additional15 min. The layers were separated, and the aqueous layer was extractedwith ethyl acetate. The combined organic layers were dried over sodiumsulfate and concentrated to yield the crude aldehyde, which was eithercarried on crude or purified via silica gel chromatography usinghexanes/ethyl acetate as eluant.

General Method 3: Suzuki Coupling of Aryl Bromides with ArylboronicAcids

ArBr+Ar′B(OR)₂→Ar—Ar′

R=H or Alkyl

A solution of 1.0 eq of an arylboronic acid (or ester) and theappropriate aryl bromide (1.0 eq) in 2:1 toluene:ethanol (0.1 Mconcentration for each reagent) was sparged with nitrogen for 15minutes. Tetrakis (triphenylphosphine)palladium (0) (0.05 eq) and 2 Maqueous sodium carbonate (3 eq) were added and the mixture was heated at85° C. for 3 h under a nitrogen atmosphere. The mixture was cooled andethyl acetate and water were added. The organic layer was washed oncewith saturated aqueous sodium carbonate, dried over sodium sulfate, andconcentrated. The residue was chromatographed on silica gel usinghexanes/ethyl acetate as eluant to yield the biaryl product.

Non-limiting examples of arylboronic acids used:[2-[[(4,5-dimethyl-3-isoxazolyl)[(2-methoxyethoxy)methyl]amino]-sulfonyl]phenyl]boronicacid (prepared as described in U.S. Pat. No. 5,612,359 and U.S. patentapplication Ser. No. 09/013,952, filed Jan. 27, 1998);2-[[N-(4,5-dimethyl-3-isoxazolyl)-N-(methoxymethyl)amino]sulfonyl]-phenylboronicacid.

General Method 4: Reduction of Aryl Aldehydes to Benzylic Alcohols UsingSodium Borohydride

ArCHO→ArCH₂—OH

Sodium borohydride (0.5 eq) was added at 0° C. to a 0.2 M solution of anaromatic aldehyde in absolute ethanol or methanol. The mixture wasallowed to warm to rt and stirred for 1-2 h. Aqueous potassiumdihydrogen phosphate solution (or dilute hydrochloric acid) was addedand the mixture was stirred for an additional 15 min. The mixture waspartially concentrated and the residue partitioned between ethyl acetateand water. The aqueous layer was extracted twice with ethyl acetate andthe combined organic extracts were dried over sodium sulfate andconcentrated. The crude benzylic alcohol was either used directly or waspurified by silica gel chromatography using hexanes/ethyl acetate aseluant.

General Method 5: Conversion of Benzyl Alcohols to Benzyl Bromides

RCH₂OH→RCH₂Br

To a 0.2 M solution of the benzyl alcohol in DMF at 0° C. was addedcarbon tetrabromide (1.5 eq) followed by triphenylphosphine (1.5 eq).The mixture was stirred at 0° C. for 4 h, diluted with 10 parts 2:1hexanes/ethyl acetate, and washed with water and brine. The solution wasdried over sodium sulfate and concentrated, and the residuechromatographed on silica gel using hexanes/ethyl acetate as eluant toyield the benzyl bromide product.

General Method 6: Hydrolysis of SEM or MEM Sulfonamide Protecting GroupsUsing Hydrochloric Acid/Ethanol

To a 0.1 M solution of a SEM- or MEM-protected N-heteroaryl sulfonamidein one volume of 95% EtOH was added an equal volume of 6N aqueous HCl,and the resulting solution was heated at reflux for 1 h. The reactionmixture was concentrated and the pH of the solution was adjusted to pH 8using aqueous sodium bicarbonate solution. It was then reacidified to pH5 with glacial acetic acid. The mixture was extracted with threeportions of ethyl acetate. The combined organic extracts were washedwith water and brine, dried over sodium sulfate, and concentrated. Theresidue was purified by reverse-phase preparative HPLC, or by silica gelchromatography using chloroform/methanol or hexanes/acetone as eluant.

General Procedure: Purification by Anion Exchange Chromatography

Anion exchange chromatography was performed on Varian SAX cartridges(acetate form, 1.5-3 g) or United Chemical Technologies CUQAX13M6-ACcartridges (acetate form, 3 g). Following a methanol rinse, thecartridge was loaded with a dichloromethane solution of crude product.Elution of impurities with dichloromethane, followed by elution of thedesired product with 1-3% TFA in dichloromethane ordichloromethane/methanol, provided the purified product.

General Procedure: Purification by Reverse-Phase Preparative HPLC

Reverse-phase preparative HPLC was performed with Shimadzu 8A liquidchromatographs using YMC S5 ODS columns (20×100, 20×250, or 30×250 mm).Gradient elution was performed with methanol/water mixtures in thepresence of 0.1% TFA. In some cases a product eluting as a TFA salt wassubsequently converted to the corresponding free base by extraction fromaqueous sodium bicarbonate or sodium carbonate solution.

Analytical HPLC Methods Employed in Characterization of Examples

Analytical HPLC was performed on Shimadzu LC10AS liquid chromatographsusing the following methods: A. Linear gradient of 0 to 100% solvent Bover 4 min, with 1 min hold at 100% B; UV visualization at 220 nmColumn: YMC S5 ODS Ballistic 4.6×50 mm Flowrate: 4 ml/min Solvent A:0.1% trifluoroacetic acid, 90% water, 10% methanol Solvent B: 0.1%trifluoroacetic acid, 90% methanol, 10% water B. Linear gradient of 0 to100% solvent B over 30 min, with 5 min hold at 100% B; UV visualizationat 254 nm Column: YMC S3 ODS 6.times.150 mm Flowrate: 1.5 ml/min SolventA: 0.2% phosphoric acid, 90% water, 10% methanol Solvent B: 0.2%phosphoric acid, 90% methanol, 10% water C. Linear gradient of 0 to 100%solvent B over 4 min, with 1 min hold at 100% B UV visualization at 220nm Column: YMC 55 ODS Ballistic 4.6×50 mm Flowrate: 4 ml/min Solvent A:0.2% phosphoric acid, 90% water, 10% methanol Solvent B: 0.2% phosphoricacid, 90% methanol, 10% water D. Linear gradient of 45 to 100% solvent Bover 2 min, with 1 min hold at 100% B; UV visualization at 220 nmColumn: Phenomenex Primesphere 4.6×30 mm Flowrate: 5 ml/min Solvent A:0.2% phosphoric acid, 90% water, 10% methanol Solvent B: 0.2% phosphoricacid, 90% methanol, 10% water E. Same conditions as (B), but with alinear gradient of 40 to 100% solvent B over 30 min, with 5 min hold at100% B F. Same conditions as (B), but with a linear gradient of 70 to100% solvent B over 30 min, with 5 min hold at 100% B G. Same conditionsas (D), but with a linear gradient of 40 to 100% solvent B over 2 min,with 1 min hold at 100% B H. Linear gradient of 0 to 100% solvent B over2 min, with 1 min hold at 100% B; UV visualization at 220 nm Column:Phenomenex Primesphere 4.6×30 mm Flowrate: 5 ml/min Solvent A: 0.1%trifluoroacetic acid, 90% water, 10% methanol Solvent B: 0.1%trifluoroacetic acid, 90% methanol, 10% water I. Same conditions as (B),but with a linear gradient of 50 to 100% solvent B over 30 min, with 5min hold at 100% B J. Same conditions as (C), but with a linear gradientof 0 to 100% solvent B over 8 min, with 1 min hold at 100% B K. Sameconditions as (D), but with a linear gradient of 0 to 100% solvent Bover 2 min, with a 1 minute hold at 100% B.

EXAMPLES Example 1

Name:4′-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-2′-formyl-N-(4,5-dimethyl-3-isoxazolyl)-[[1,1′-biphenyl]-2-sulfonamide]A.4′-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-2′-formyl-N-(4,5-dimethyl-3-isoxazolyl)-N-(2-methoxyethoxymethyl)[1,1′-biphenyl]-2-sulfonamide

Palladium catalyzed Suzuki coupling of4-bromo-3-(ethoxymethyl)benzaldehyde and[2-[[(4,5-dimethyl-3-isoxazolyl)[(2-methoxyethoxy)methyl]amino]sulfonyl]phenyl]boronicacid was performed according to General Method 3 to affordN-(4,5-dimethyl-3-isoxazolyl)-4′-(ethoxycarbonyl)-2′-(formyl)-N-((methoxyethoxy)methyl)[1,1′-biphenyl]-2-sulfonamide(81%) following silica-gel chromatography.

B. 4′-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-2′-formyl-N-(4,5-dimethyl-3-isoxazolyl)-[1,1′-biphenyl]-2-sulfonamide

Treatment ofN-(4,5-dimethyl-3-isoxazolyl)-4′-(ethoxycarbonyl)-2′-(formyl)-N-((methoxyethoxy)methyl)[1,1′-biphenyl]-2-sulfonamidewith 6N aqueous hydrochloric acid according to General Method 6 toremove the MEM protecting group provided the title compound (85%):Rf=0.38, 5% MeOH in methylene chloride.

Example 2

4′-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-N-(4,5-dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)-[1,1′-biphenyl]-2-sulfonamidewas synthesized by combinations of the General Methods.

Name:4′-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-N-(4,5-dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)-[1,1′-biphenyl]-2-sulfonamide

Starting Material: X—Br or OMs

General Methods Applied (yield, %): General Method 1, EtOH (77); GeneralMethod 2 (80); General Method 3 (70); General Method 4 (98); GeneralMethod 5 (80); General Method 1 (83); General Method 6 (86)

M/z (MH)⁺: 593

HPLC % Purity: >98

HPLC retention time, min (HPLC method): 18.75 (E)

Example 34′-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-N-(4,5-dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)[1,1′-biphenyl]-2-sulfonamide[crystalline]

Alternative Synthesis of4′-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-N-(4,5-dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)[1,1′-biphenyl]-2-sulfonamideStep A. Ethyl 4-bromo-3-(bromomethyl)benzoate

Ethyl 4-bromo-3-methylbenzoate (110 g, 450 mmol.) was treated with NBSaccording to the procedure of Example 5. Silica gel chromatography withhexanes/ethyl acetate as eluant provided ethyl4-bromo-3-(bromomethyl)benzoate (91 g, 62%) as a white solid.

Step B. Ethyl 4-bromo-3-(ethoxymethyl)benzoate

A solution of ethyl 4-bromo-3-(bromomethyl)benzoate (89 g, 280 mmol.) ina mixture of ethanol (300 mL) and DMF (50 mL) was treated at 0° C. withsodium ethoxide (135 mL of a 21% solution in ethanol). The mixture wasallowed to warm to rt and was stirred for 16 h. The ethanol wasevaporated under reduced pressure. Ethyl acetate was added to theresidue and the mixture was washed with water and brine. The organiclayer was dried over sodium sulfate and concentrated, and the residuewas chromatographed on silica gel using hexanes/ethyl acetate as eluantto provide Ethyl 4-bromo-3-(ethoxymethyl)benzoate (67 g, 84%) as aslightly yellow oil.

Step C.N-(4,5-Dimethyl-3-isoxazolyl)-4′-(ethoxycarbonyl)-2′-(ethoxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamide

Ethyl 4-bromo-3-(ethoxymethyl)benzoate (32 g, 100 mmol) was subjected toSuzuki coupling with2-[[N-(4,5-dimethyl-3-isoxazolyl)-N-(methoxymethyl)amino]sulfonyl]-phenylboronicacid according to General Method 3. Silica gel chromatography usinghexanes/ethyl acetate as eluant providedN-(4,5-Dimethyl-3-isoxazolyl)-4′-(ethoxycarbonyl)-2′-(ethoxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamide(52 g) as a yellow oil, contaminated with by-products deriving from theboronic acid.

Step D.N-(4,5-Dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)-4′-(hydroxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamide

N-(4,5-Dimethyl-3-isoxazolyl)-4′-(ethoxycarbonyl)-2′-(ethoxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamide (entire sample) was treated with DIBAL-Haccording to the following procedure:

A solution ofN-(4,5-Dimethyl-3-isoxazolyl)-4′-(ethoxycarbonyl)-2′-(ethoxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamide(0.3 mmol.) in THF (5 mL) is treated with DIBAL-H (0.53 mL of a 1.5 Msolution in toluene, 0.8 mmol.) at −78° C. The temperature was allowedto rise to −25° C. and the mixture is stirred for 2 h. Saturated aqueousammonium chloride is added to the cooled reaction mixture, followed byextraction with ethyl acetate. The combined organic layers are driedover sodium sulfate and concentrated to provideN-(4,5-Dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)-4′-(hydroxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamideas a crude yellow oil.

Step E.4′-(Bromomethyl)-N-(4,5-dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamide

N-(4,5-Dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)-4′-(hydroxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamide (entire sample) was converted tothe corresponding bromide according to General Method 5. Silica gelchromatography using hexanes/ethyl acetate as eluant provided4′-(Bromomethyl)-N-(4,5-dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamide](38g, purity estimated to be 83% by ¹H NMR) as a light yellow oil.

Step F. 4′-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-N-(4,5-dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamide

4′-(Bromomethyl)-N-(4,5-dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamide] (entire sample) was used to alkylate2-butyl-1,3-diazaspiro[4.4]non-1-en-4-one according to General.Method 1. The crude residue was chromatographed on silica gel usinghexanes/ethyl acetate/triethylamine as eluant to provide4′-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl-N-](4,5-dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamide(32 g, 53% from Step B) as a slightly yellow oil.

Step G.4′-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-N-(4,5-dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)[1,1′-biphenyl]-2-sulfonamide

The MOM protecting group of4′-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-N-(4,5-dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)-N-(methoxymethyl)[1,1′-biphenyl]-2-sulfonamide (32 g, 53 mmol.) was removed according toGeneral Method 6. The crude product was purified by silica gelchromatography using hexanes/ethyl acetate/acetic acid as eluant toprovide the title compound (26 g, 88%) as an amorphous foam: MS n/e 593(ESI+ mode); HPLC retention time 18.75 min (HPLC method E); HPLCpurity>96%.

Example 4

Step H.4′-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-N-(4,5-dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)[1,1′-biphenyl]-2-sulfonamide

Crystallization

The amorphous4′-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-N-(4,5-dimethyl-3-isoxazolyl)-2′-(ethoxymethyl)[1,1′-biphenyl]-2-sulfonamide(1 g) was dissolved in 5 mL of isopropanol then 5 mL of water was addedto the mixture dropwise and the mixture was warmed up to 40° C. toprovide a clear solution. The solution was allowed to stand at roomtemperature and the white crystals thus obtained were filtered andwashed with a small amount of 2:1 mixture of isopropanol/water and driedto give 0.87 g of a white crystalline solid. mp: 148° C.

Example 5 General Method 1: Benzylic Bromination UsingN-Bromosuccinimide

To a 0.4 M solution of a methyl-substituted aromatic compound, such asethyl 4-bromo-3-methylbenzoate, in carbon tetrachloride was addedN-bromosuccinimide (1.05 eq) and benzoyl peroxide (0.03 eq), and themixture was heated at reflux for 8-16 h. The mixture was cooled andfiltered and the filtrate concentrated. The residue was purified bytrituration with 3:1 hexanes/ethyl acetate, or by silica gelchromatography using hexanes/ethyl acetate as eluant to provide themono-brominated product, such as ethyl 4-bromo-3-(bromomethyl)benzoate.

Example 6 200 mg and 400 mg Strength Tablets for Oral AdministrationUsing Low Shear Granulation

Two formulations utilizing microcrystalline cellulose versus a mixtureof microcrystalline cellulose and lactose were processed using low sheargranulation. The compositions of Formulation B and Formulation C areshown in Tables 1 and 2. 200 mg and 400 mg strength tablets were madeusing a low shear wet granulation method of manufacture with a 57% drugload.

TABLE 1 Compositions of 200 mg Strength Tablets Formulation CFormulation B Amount Compo- Amount Compo- mg/ sition mg/ sitionINGREDIENT tablet % by weight tablet % by weight Intragranular Compoundof formula 200.0 57.1 200.0 57.1 I (API) Microcrystalline 102.4 29.376.43 21.8 Cellulose, NF Avicel PH 101 Lactose Monohydrate — — 38.2210.9 316 (Fast Flow) Sodium Lauryl Sulfate, — — 3.50 1.0 NFCroscarmellose Sodium 8.75 2.5 8.75 2.5 (Ac-Di-Sol)Hydroxypropylcellulose, 10.5 3.0 10.5 3.0 EXF Poloxamer 188, NF 17.5 5.0— — Extragranular Croscarmellose Sodium 8.75 2.5 8.75 2.5 (Ac-Di-Sol)Colloidal Silicone 0.35 0.1 0.35 0.1 Dioxide, NF (Cab-O-Sil M5P)Magnesium Stearate, 1.75 0.5 3.50 1.0 NF (Vegetable Grade) Total 350 350

TABLE 2 Compositions of 400 mg Strength Tablets Formulation CFormulation B Amount Compo- Amount Compo- mg/ sition mg/ sitionIngredient tablet % by weight tablet % by weight Intragranular Compoundof formula 400.0 57.1 400.0 57.1 I (API) Microcrystalline 204.8 29.3152.86 27.8 Cellulose, NF Avicel PH 101 Lactose Monohydrate — — 76.4410.9 316 (Fast Flow) Sodium Lauryl Sulfate, — — 7.0 1.0 NFCroscarmellose Sodium 17.5 2.5 17.5 2.5 (Ac-Di-Sol)Hydroxypropylcellulose, 21.0 3.0 21.0 3.0 EXF Poloxamer 188, NF 35.0 5.0— — Extragranular Croscarmellose Sodium 17.5 2.5 17.5 2.5 (Ac-Di-Sol)Colloidal Silicone 0.7 0.1 0.7 0.1 Dioxide, NF (Cab-O-Sil M5P) MagnesiumStearate, 3.5 0.5 7.0 1.0 NF (Vegetable Grade) Total 700 700

The formulations were granulated with a Kitchen Aid planetary type mixerusing milli-Q water as the granulation liquid. Formulation C wasgranulated with a total of 84 grams of water at an addition rate of 14grams per minute. Formulation B was granulated with a total of 94 gramsof water with an addition rate of 14 grams per minute. Both granulationswere tray dried in a 60° C. vented oven for approximately 15 hours. Thedried granulations were milled using a 197S Quadro Comil equipped with a050G grater screen, a round impeller, and a 0.175″ spacer at 20% Comilspeed. The extragranular excipients were adjusted based on the percentyield. Each formulation was bag blended for 5 minutes before thelubrication was added and blended an additional 1 minute after thelubrication was added.

Physical properties of the blends were determined (bulk density and LODmoisture content). The blend was compressed on a Piccola BD tablet pressset up with a gravity feed frame and hopper at a speed of 36 rpm.Pre-compression, main compression and ejection forces were monitoredusing “The Director” software, written by SMI Inc. Tablet weights of 350mg and 700 mg were targeted to produce 200 and 400 mg strength tabletsrespectively from each formulation. The 350 mg tablets (200 mg strength)were compressed with 0.2500″×0.5000″ capsule shaped standard concavetooling and the 700 mg tablets (400 mg strength) were compressed with0.3300×0.7100 oval standard concave tooling. A range of tablethardnesses were targeted to determine the robustness of the formulationsand tablet thickness was recorded. Tablet weight, hardness, thicknessand friability were monitored in-process.

The low shear granulation process succeeded in moderately densifying theformulations and tray drying brought the wet granulation to anacceptably low moisture content as indicated by the density and LODresults as shown in Table 3.

TABLE 3 Bulk Density and LOD Results for Formulation C and Formulation BBulk density of LOD of wet LOD after tray Formulation granulation (g/ml)granulation (%) drying (%) Formulation C 0.39 23.61 1.10 Formulation B0.40 26.90 1.39

Both tablet strengths of Formulation C and Formulation B performed wellon the rotary tablet press with regard to flow and compressibilityresulting in stable tablet weights and acceptable tablet hardness andlow friability (Tables 4 and 5). Both tablet formulations disintegratedrapidly in 0.1 N HCl, but the Formulation B disintegrated faster thanFormulation C (Table 5). Formulation C experienced tablet stickingissues during the compression trials. Further, addition of magnesiumstearate was studied.

TABLE 4 Tablet Physical Testing for Formulation C and Formulation BAverage tablet Average tablet Average tablet weight (mg) thickness (in)hardness (kp) Formulation [n = 10] [n = 10] [n = 10] Formulation C 200mg 351.5 0.196 13.8 strength tablets Formulation C 400 mg 697.6 0.26912.1 strength tablets Formulation B 200 mg 348.0 0.202 12.7 strengthtablets Formulation B 400 mg 716.7 0.287 9.6 strength tablets

TABLE 5 Disintegration Results for 200 mg and 400 mg Strength Tabletsfor Formulation C and Formulation B Disinte- Disinte- Target grationgration Friability (%) Hardness (0.1N HCl) (0.1N HCl) (100 Drops) (kp)Initial (min) Final (min) [n = 10] Formulation 200 mg StrengthFormulation C 8-9 3.15 4.35 0.04 Formulation B 8-9 1.32 1.78 0.12Formulation C 15 6.93 9.70 No data Formulation B 15 2.57 4.95 No dataFormulation 400 mg Strength Formulation C 8-9 No data No data 0.10Formulation B 8-9 No data No data 0.20

HPLC-backend analysis was performed on the dissolution samples.Dissolution test results indicated that Formulation C had asubstantially faster dissolution rate than Formulation B in the 0.1 NHCl dissolution media at both 50 and 60 rpm paddle speed (Table 6).

TABLE 6 Dissolution Results in 0.1N HCL Dissolution Media for 400 mgStrength Tablets: Formulation C and Formulation B 400 mg strength highhardness % Dissolved vs Time (min) (~14 kp) tablet sample 0 10 20 30 4560 Formulation B, 50 rpm 0 37 53 61 67 71 Formulation B, 60 rpm 0 44 6575 83 87 Formulation C, 50 rpm 0 56 73 79 84 87 Formulation C, 60 rpm 062 82 87 91 94

Assay and related substance results for Formulation C and Formulation Bare shown in Table 7.

TABLE 7 Assay and Related Substances Results for Formulation C andFormulation B Tablets Assay potency % Impurity versus Relative retentiontime % label RRT RRT RRT RRT RRT RRT RRT Total Sample tablet claim 0.850.88 0.96 0.98 1.02 1.03 1.21 impurities Formulation C 99.5 0.06 0.130.05 0.06 0.07 0.05 0.37 0.75 Formulation B 99.4 0.06 0.13 0.05 0.060.07 0.05 0.38 0.77

Example 7 200 mg and 400 mg Strength Tablets for Oral AdministrationUsing High Shear Wet Granulation

In Example 6, Formulation C of a microcrystalline cellulose basedcomposition including Poloxamer 188 was observed to have a substantiallyfaster drug release profile compared to Formulation B of amicrocrystalline cellulose/lactose combination formulation (Table 6).Formulation C needed an increase in lubricant due to sticking issuesseen in compression trials. In this example, the amount of magnesiumstearate was increased in Formulation C to provide Formulation D. Also,a new experimental design was created to evaluate the super disintegrantsodium starch glycolate in addition to croscarmellose sodium to provideFormulation E thereby further increasing disintegration/dissolutionrates. A high shear wet granulation was substituted for the low sheargranulation to improve the dissolution rate and provide a manufacturingmethod more modern and suitable for future scale up manufacture.

Formulation D is a microcrystalline cellulose based formulation withPoloxamer 188 including a 0.5% increase in lubrication and addition ofcroscarmellose sodium. Formulation E is a similar formulation usingsodium starch glycolate instead of croscarmellose sodium disintegrant.Formulation A is an additional formulation including microcrystallinecellulose, croscarmellose sodium with sodium lauryl sulfate in place ofPoloxamer 188. Compositions of Formulations D, E, and A are shown inTables 8 and 9. Dose proportional 200 mg and 400 mg strength tabletswere prepared with the ingredients in the amount as shown in Tables 8and 9.

The formulations were granulated using the high shear wet granulation inthe 10 liter Niro PP 1 high shear granulator equipped with a 7.5 literinsert. Milli-Q water was used as the binder solution. The batch sizefor each formulation was 750 grams. The intra-granular materials weremixed in the granulator for 3 minutes at 300 rpm impeller speed prior togranulation. Formulation D granulation required 251 grams of water at aspray rate of 41.8 g/minute. Formulation A granulation required 322grams of water at a spray rate of 41.8 g/minute. Formulation E required311 grams of water at a spray rate of 52 g/minute. The impeller speedwas set at 305 rpm and chopper set on high speed during granulation. Thegranulation end point was qualitatively determined by the “snowball”method (first test) and the granulations were mixed an additional 5minutes at 300 rpm with no chopper. Each formulation was dried with theNiro Aeromatic MP-1 fluid bed dryer at an inlet air temperature of 65°C. to an LOD moisture content of less than 2.0%. The dried granulationswere milled using a 197S Quadro Comil equipped with a 0.050″ graterscreen, a round impeller and a 0.175″ spacer at 20% Comil speed.

TABLE 8 Compositions of 200 mg Strength Tablets for Formulation D,Formulation A and Formulation E Formulation D Formulation A FormulationE Amount Composition Amount Composition Amount Composition Ingredientmg/tablet % by weight mg/tablet % by weight mg/tablet % by weightIntragranular Compound of formula I 200.0 57.1 200.0 57.1 200.0 57.1Microcrystalline Cellulose, 100.65 28.8 114.65 32.8 100.65 28.8 NFAvicel PH 101 Sodium Lauryl Sulfate, NF — — 3.50 1.0 — — CroscarmelloseSodium 8.75 2.5 8.75 2.5 — — (Ac-Di-Sol) Hydroxypropylcellulose, 10.503.0 10.50 3.0 10.50 3.0 EXF Poloxamer 188, NF 17.5 5.0 — — 17.5 5.0Sodium Starch Glycolate — — — — 8.75 2.5 (EXPLOTAB ©) ExtragranularSodium Starch Glycolate — — — — 8.75 2.5 (EXPLOTAB ©) CroscarmelloseSodium 8.75 2.5 8.75 2.5 — — (Ac-Di-Sol) Colloidal Silicone Dioxide,0.35 0.1 0.35 0.1 0.35 0.1 NF (Cab-O-Sil M5P) Magnesium Stearate, NF3.50 1.0 3.50 1.0 3.50 1.0 (Vegetable Grade) Total 350 350 350

TABLE 9 Compositions of 400 mg Strength Tablets for Formulation D,Formulation A, and Formulation E Formulation D Formulation A FormulationE Amount Composition Amount Composition Amount Composition Ingredientmg/tablet % by weight mg/tablet % by weight mg/tablet % by weightIntragranular Compound of formula I 401.8 57.4 401.8 57.4 401.8 57.4Microcrystalline Cellulose, 201.3 28.8 229.3 32.8 201.3 28.8 NF AvicelPH 101 Sodium Lauryl Sulfate, NF — — 3.50 1.0 — — Croscarmellose Sodium17.5 2.5 17.5 2.5 — — (Ac-Di-Sol) Hydroxypropylcellulose, 21.0 3.0 21.03.0 21.0 3.0 EXF Poloxamer 188, NF 35.0 5.0 — — 35.0 5.0 Sodium StarchGlycolate — — — — 17.5 2.5 (EXPLOTAB ©) Extragranular Sodium StarchGlycolate — — — — 17.5 2.5 (EXPLOTAB ©) Croscarmellose Sodium 17.5 2.517.5 2.5 — — (Ac-Di-Sol) Colloidal Silicone Dioxide, 0.7 0.1 0.7 0.1 0.70.1 NF (Cab-O-Sil M5P) Magnesium Stearate, NF 7.0 1.0 7.0 1.0 7.0 1.0(Vegetable Grade) Total 700 700 700

The extragranular excipients were adjusted per the yield and added toeach example granulation. The mixture was bag blend for 5 minutes.Magnesium stearate was layered into the mixture and the mixture wasblended for an additional 3 minutes. The final blends were compressed ona Piccola ED tablet press using the paddle feeder feed frame at a pressspeed of 36 rpm. The 350 mg tablets (200 mg strength) were compressedwith 0.2500″×0.5000″ capsule standard concave tooling and the 700 mgtablets (400 mg strength) were compressed with 0.3300×0.7100 ovalstandard concave tooling. A tablet hardness of 15 KP was targeted forthe formulations.

The physical properties of the final blends for Formulation D, andFormulation A, showed little difference in relation to bulk density, tapdensity, angle of repose, compressibility or particle size distribution(Tables 10 and 11). All examples exhibited acceptable flow as indicatedby the angle of repose, compressibility and Flodex values (Table 10).All formulations exhibited low moisture as shown in Table 11.Formulation E was substantially denser than the other two Formulationsand was substantially larger in its particle size distribution as shownin Table 11. The Formulation E wet granulation process produced large,well defined granules. Large hard granules produced during granulationresulted in significantly longer milling times than seen in the otherexamples. Some dried spheres, granules and particles could not be milledand were discarded.

TABLE 10 Bulk Density and Tap Density Results Bulk Tap Flodex Compress-Density Density Angle of Orifice ibility Formula (g/ml) (g/ml) Repose(°) (mm) (%) Formulation D 0.47 0.61 29.7 — 16.6 Formulation A 0.48 0.6030.1 — 16.6 Formulation E 0.52 0.64 29.4 8 16.7 Formulation G 0.57 0.6731.8 5 14.3 Formulation F 0.49 0.63 32.7 7 16.7 Formulation D 0.45 0.5634.6 — 16.7 (larger API particle size)

TABLE 11 Particle Size Data of Final Blends Formulation For- For- For-For- For- D (larger mula- mula- mula- mula- mula- API particle tion tiontion tion tion size) 100 mg Micron Sieve D A E G F and 400 mg 850 20 2.12.0 11.3 6.1 2.9 3.9 425 40 13.5 13.1 54.6 31.3 15.3 13.9 250 60 10.412.1 13.4 32.3 10.6 6.9 180 80 7.3 9.1 4.1 12.1 6.7 5.9 106 140 10.412.1 4.1 7.1 9.6 17.8 45 325 44.8 33.3 7.2 6.1 37.5 43.6 <45 Pan 11.518.2 5.2 5.0 17.3 7.9

LOD results for Formulations A, D, E, G and F are shown in Table 12.

TABLE 12 LOD Results Formulation LOD after Fluid Bed Drying (%)Pre-Granulation 1.90 Formulation D 1.50 Formulation A 1.20 Formulation E1.00 Formulation G 0.75 Formulation F 1.00

All the formulation blends performed well on the tablet press (Table 13)and produced high quality, consistent, hard tablets with low friability.The increased lubricant in Formulation D eliminated the tablet stickingissues that were seen earlier in Formulation C. The tabletdisintegration time at 49.70 minutes for Formulation E was significantlylonger than the tablet disintegration time seen in the other examples(Table 14).

TABLE 13 Tablet Physical Testing Data for 200 mg and 400 mg StrengthTablets Average Average Average Tablet Tablet Tablet Tablet FriabilityAverage Weight Thickness Hardness (100 drops) Average Pre- AverageEjection (mg) (in) (kp) (%) Compression Compression Force Formulation [n= 10] [n = 10] [n = 10] [n = 10] Force (N) Force (KN) (N) Formulation350.1 0.192 14.6 0.1 22.9 7.0 123 D 200 mg Formulation 700.6 0.254 15.00.1 22.9 8.5 130 D 400 mg Formulation 358.1 0.199 15.2 0.1 24.1 6.7 147A 200 mg Formulation 704.8 0.264 14.9 0.2 20.1 8.0 160 A 400 mgFormulation 352 0.190 14.3 0.0 24.0 8.7 149.9 E 200 mg Formulation 7290.258 15.8 0.1 8.4 E 400 mg Formulation 358.7 0.188 12.0 0.1 25.9 14.7188.9 G 200 mg Formulation 707.9 0.245 11.3 0.3 G 400 mg Formulation350.9 0.191 14.0 0.0 27.3 7.7 169.0 F 200 mg Formulation 175.5 0.18910.1 0.2 D (larger API particle size) 100 mg Formulation 704.4 0.26716.1 0.0 D (larger API particle size) 400 mg

TABLE 14 Disintegration Results for 200 mg Strength TabletsDisintegration Disintegration Formulation 200 mg Target (0.1N HCl) (0.1NHCl) Strength Hardness (kp) Initial (min) Final (min) Formulation D LS8-9 3.15 4.35 Formulation B LS 8-9 1.32 1.78 Formulation D LS 15 6.939.70 Formulation B LS 15 2.57 4.95 Formulation A HS 15 0.93 1.17Formulation D HS 15 15.13 17.0 Formulation E HS 15 41.48 49.70Formulation G HS 12 30.25 34.35 Formulation F HS 14 11.57 15.00 LS = lowshear granulation process; HS = high shear granulation process.

The Poloxamer 188 surfactant (Formulation D) outperformed the sodiumlauryl sulfate surfactant (Formulation A) with respect to dissolution.Both Formulation A and Formulation D outperformed Formulation E. Thedissolution profile for the 400 mg strength tablet, Formulation A showeda 77% drug release in 30 minutes and an 89% drug release in 60 minuteswhile the dissolution profile for the 400 mg strength tablet ofFormulation D, showed a 92% drug release in 30 minutes and a 96% drugrelease in 60 minutes (Table 15).

TABLE 15 Dissolution Results for 400 mg Strength Tablets Formulation A,D and E in 0.1N HCl at 60 rpm 400 mg High Hardness % Dissolved vs Time(min) (~14 kp) Tablets sample 0 10 20 30 45 60 75 Formulation A 0 52 7077 84 89 93 Formulation D 0 51 86 92 94 96 98 Formulation E 0 14 27 3853 64 80

The 200 mg strength Formulation A tablets had a faster dissolutionrelease than the 400 mg strength tablets as shown in Table 16.

TABLE 16 Dissolution Results for Formulation A 400 mg and 200 mgStrength Tablets % Dissolved vs Time (min) Tablets Sample 0 10 20 30 4560 75 Formulation A 400 mg strength tablets 0 52 70 77 84 89 93Formulation A 200 mg strength tablets 0 67 83 90 95 96 99

Both Formulation D and Formulation A were shown to have a high degree ofpotency with low related substances. A short term stability evaluationwas performed on the 200 mg strength Formulation D and Formulation Atablets in packaged and open container conditions. The packaged tabletswere placed in HDPE bottles with induction sealed closures and subjectedto accelerated stability conditions at 50° C. (ambient humidity). Theopen container tablets were place in open bottles at 40° C./75% RH(relative humidity). The tablets were assayed at 2 and 4 week's time.The short term stability study indicated that both Formulation D andFormulation A were chemically stable formulations. 200 mg strengthtablets for both Formulation D and Formulation A remained potent andwith no significant increase in related substances for at least 4 weeksat 40° C./75% RH in open containers. The 50° C. closed container tabletswere stable for 4 weeks with only some minor degradation seen in theFormulation D sample at the 4-week time point. No increase indegradation was seen in Formulation A.

TABLE 17 Assay and Related Substance Results for 4 week Short TermStability Study for 200 mg Strength Tablets for Formulation D andFormulation A Formulation D Formulation A Total Total Related RelatedConditions/ Assay Substance Assay Substance Time Point % LC (%) % LC (%)Initial 99.1 0.74 100.5 0.73 40° C./75% RH 100.0 0.63 102.4 0.55 Open 2weeks 40° C./75% RH 99.9 0.70 100.7 0.73 Open 4 weeks 50° C. Closed 2weeks 100.3 0.66 101.4 0.57 50° C. Closed 4 weeks 97.4 1.00 100.0 0.72

From the experimentation performed, particularly the dissolution, it wasobserved that the Poloxamer 188 was a good surfactant and thatcroscarmellose sodium was a good disintegrant. High shear wetgranulation proved to be an effective method of manufacture. Theformulations using low shear wet granulation had less advantages incomparison with the formulations manufactured with the high shear wetgranulation. The dissolution testing provided some such evidence of thedifferences between the two methods.

Example 8 Compression Studies Evaluating Formulation a and Formulation C

Tablet compression studies were performed on Formulation D, andFormulation A. Compression was performed on the Piccola BD Tablet Pressat a speed of 36 rpm. Five main compression forces ranging from 3 to 10kN was targeted to stress the formulation. Tablet weight (mg), tabletedge thickness, and tablet edge thickness (in) was measured.

The compression studies showed that all formulations were highlycompressible. Formulation A started to reach its maximum hardness around21 kp at 10.0 kN whereas Formulation D reached it maximum hardnessaround 16.3 kp at 9.9 kN. It was also seen in the compression studiesthat Formulation A required more force than Formulation D to achieve thesame tablet thickness. Formulation D had slightly lower ejection forces.Formulation A provided higher hardness in comparison to Formulation D.

Example 9 Optimization of High Shear Granulation

The sieve analysis of the final blend of Formulation D indicated a highlevel of fines in the granulation (˜50%). To further optimizeformulation D, and reduce the level of fines in the granulation,experimentation was undertaken to possibly improve the high sheargranulation process by increasing the amount of granulation fluid from25% w/w (Formulation F) to 30% w/w (Formulation G). Formulations used inthe optimization experimentation are presented in Table 18.

TABLE 18 Granulation Study of 200 mg Strength Formulations Formulation GFormulation F Amount Compo- Amount Compo- mg/ sition % mg/ sition %INGREDIENT tablet by weight tablet by weight Intragranular Compound ofFormula I 200.0 57.1 200.0 57.1 Microcrystalline Cellulose, 100.65 28.8100.65 28.8 NF Avicel PH 101 Sodium Lauryl Sulfate, NF — — — —Croscarmellose Sodium 8.75 2.5 8.75 2.5 (Ac-Di-Sol)Hydroxypropylcellulose, 10.5 3.0 10.5 3.0 EXF Poloxamer 188, NF 17.5 5.017.5 5.0 Sodium Starch Glycolate — — — — (EXPLOTAB ©) ExtragranularSodium Starch Glycolate — — — — (EXPLOTAB ©) Croscarmellose Sodium 8.752.5 8.75 2.5 (Ac-Di-Sol) Colloidal Silicone 0.35 0.1 0.35 0.1 Dioxide,NF (Cab-O-Sil M5P) Magnesium Stearate, NF 3.50 1.0 3.50 1.0 (VegetableGrade) Total 350 350

Both batches of pre-granulation excipients were blended using a 1 QuartV Blender. The compound of formula I (API) was sandwiched between theblended excipients in the Niro PP-1 blended for 5 minutes with chopperoff. The formulations were granulated by high shear granulation usingthe Niro PP1, equipped with a 7.5 liter insert. Milli-Q water was usedas the granulation solution. The batch size was 1000 grams forFormulation G and 950 g for Formulation F due to limited API.Formulation G was granulated using 413 grams of water at a spray rate of68.6 g/minute and Formulation F was granulated using 305 grams of waterat a spray rate of 50.9 g/minute. The dried granulation was milled usinga 197S Quadro Comil equipped with a 0.050″ grater screen, a roundimpeller and a 0.175″ spacer at 20% Comil speed. The extra-granularexcipients were adjusted based on the percent yield of the final milledgranulation.

The granulation was blended in a 2 quart V blender for 5 minutes afterthe addition of the croscarmellose and the colloidal silicon dioxide.The blend was blended an additional 3 minutes after the addition of themagnesium stearate. The blend was compressed on a Piccola BD Press usingthe paddle feed frame and hopper at a speed of 36 rpm. The 350 mgtablets were compressed with 0.2500″×0.5000″ capsule standard concavetooling and the 700 mg tablets were compressed with 0.3300×0.7100 ovalstandard concave tooling.

The 200 mg strength tablets processed with 25% granulating fluid(Formulation F) were packaged 30 tablets per bottle in 50 cc HDPEbottles with induction sealed closures and placed on stability at 25°C./25% RH and 40° C./75% RH for up to 3 months along with the 200 mgstrength Formulation A tablets.

The granulation of Formulation F had a finer, powdery appearance thanFormulation G formed well defined granules. The fines were drasticallyreduced in Formulation G using 30% granulation fluid during processingas compared to Formulation F using 25% granulation fluid duringprocessing in the sieve analysis of the final blends, and particle sizedistribution of the 30% granulation (Formulation G) was substantiallylarger than the 25% granulation (Formulation F) (Table 11). The finalblends from both granulations were able to produce robust, good qualitytablets with consistent, weight hardness and thickness and lowfriability (Table 12). Assay and related substance indicated no problemwith potency or impurity. Disintegration time of the tablets roughlydoubled with the increased 30% granulation from −15 minutes to −30minutes (Table 14). Dissolution was also markedly slower with the 30%granulation tablets as compared to the 25% granulation tablets (Table19).

TABLE 19 Dissolution of 200 mg Strength and 400 mg Strength GranulationStudy Tablets Made with 25% versus 30% Granulation Fluid % Dissolved vsTime (min) Tablets Sample 0 10 20 30 45 60 75 Formulation F 200 mgstrength 0 52 70 77 84 89 93 tablets Formulation G 200 mg strength 0 921 34 51 66 80 tablets Formulation F 400 mg strength 0 51 86 95 99 100102 tablets Formulation G 400 mg strength 0 11 25 37 52 60 73 tablets

Example 10 Stability Study of Formulation F and A

Formulation F and Formulation A 200 mg strength tablets were packagedand placed on stability. The tablets were packaged 30 tablets per bottlein 50 cc HDPE bottles with induction sealed closures.

Both Formulation F and Formulation A were demonstrated to be stable forat least three months at 25° C./60% RH and 40° C./75% RH with regard toappearance, assay, related substances, dissolution, and moisturecontent. There was no indication of any instability (Table 20).

TABLE 20 Stability Data for Formulation F 200 mg Strength Tablets TP#TP# 64627 TP# 64627 TP# 64512 TP# 64730 Conditions/ 25481 Assay % LCTotal Related Dissolution % Water Time point Appearance N = 2 Substance(%) N = 6 N = 2 Initial Conforms* 100.0 0.61 30 min-91 1.6 60 min-98 25°C./60% RH Conforms* 100.5 0.53 30 min-92 1.5 1 month 60 min-95 25°C./60% RH Conforms* 101.8 0.61 30 min-89 2.1 2 month 60 min-97 25°C./60% RH Conforms* 100.1 0.53 30 min-87 2.1 3 month 60 min-95 40°C./75% RH Conforms* 100.3 0.59 30 min-88 1.5 1 month 60 min-93 40°C./75% RH Conforms* 100.1 0.61 30 min-89 2.1 2 month 60 min-94 40°C./75% RH Conforms* 99.8 0.71 30 min-89 2.1 3 month 60 min-95 *White tooff-white, convex, oblong tablets with sides scored on one sides,contained in white HDPE bottle with foil seal (12.7 mm × 6.4 mm × 5.1 mmthick)

TABLE 21 Stability Data for Formulation A 200 mg Strength Tablets TP#64627 TP# 64627 Total Related TP# 64512 TP# 64730 Conditions/ TP# 25481Assay % LC Substance Dissolution % Water Time point Appearance N = 2 (%)N = 6 N = 2 Initial Conforms* 100.5 0.73 30 min-90 2.1 60 min-96 25°C./60% RH Conforms* 101.6 0.67 30 min-87 2.2 1 month 60 min-96 25°C./60% RH Conforms* 101.7 0.80 30 min-88 2.9 2 month 60 min-95 25°C./60% RH Conforms* 101.0 0.73 30 min-86 2.4 3 month 60 min-94 40°C./75% RH Conforms* 102.7 0.62 30 min-84 2.2 1 month 60 min-97 40°C./75% RH Conforms* 101.0 0.72 30 min-87 3.0 2 month 60 min-93 40°C./75% RH Conforms* 100.4 0.69 30 min-87 2.6 3 month 60 min-93 *White tooff-white, convex, oblong tablets with sides scored on one sides,contained in white HDPE bottle with foil seal (12.7 mm × 6.4 mm × 5.1 mmthick)

Example 11 Development of 100 mg Strength Formulation D Tablet andEvaluation of the Effect of API Particle Size

The high shear granulation Formulation D was used to explore the doseproportional 100 mg strength tablet. The high shear granulationprocedure utilizing the 25% granulation fluid was employed. In addition,the effect of utilizing a larger particle sized API (the compound offormula I) was evaluated by manufacturing 400 mg strength tablets aswell as the 100 mg strength tablets from a common blend using a largerparticle sized API to compare against previous batches of 400 mgstrength tablets that used the smaller article size API.

100 mg strength tablets were made using a high shear granulationprocedure utilizing 25% granulation fluid with 57% drug load. Tabletweights of 175 mg were targeted to produce 100 mg strength tablets fromFormulation D using API with larger particle size (Table 22).

TABLE 22 Composition of 100 mg Strength Tablets for Formulation D(Larger API Particle Size) Formulation D (larger API Particle Size)Amount Composition Ingredient mg/tablet % by weight Intragranular API(Compound of formula I) 100.0 57.1 Microcrystalline Cellulose, NF 50.3328.8 Avicel PH 101 Croscarmellose Sodium (Ac-Di-Sol) 4.38 2.5Hydroxypropylcellulose, EXF 5.25 3.0 Poloxamer 188, NF 8.75 5.0Extragranular Croscarmellose Sodium (Ac-Di-Sol) 4.38 2.5 ColloidalSilicone Dioxide, NF 0.175 0.1 (Cab-O-Sil M5P) Magnesium Stearate, NF1.75 1.0 (Vegetable Grade) Total 175

The assay value used to determine the adjusted weight of the compound ofFormula I (API) was 98.80%. The total amount of compound of Formula Icalculated for the batch was 578.4 grams for a 1 kg batch size. Theexcipients were bag blended and then used to sandwich the compound ofFormula I into the PP-1 high shear granulator. The materials werecharged to the PP-1 and premixed with impeller set on 306 rpm with nochopper for 3 minutes. Granulation was performed with the impeller speedset at 306 rpm with chopper speed on high. After the granulationevaluation, it was determined that additional water was needed. A totalof 57.8 g of additional water was added to the granulation for a totalwater addition of 378.8 g at a rate of 53.5 g/min or 28% granulationfluid addition. The granulation was dried to an LOD of 0.7% in the MP-1fluid bed with an inlet air temperature of 65° C. The granulation wasmilled using a 197S Quadro Comil equipped with a 0.050″ grater screenusing a 150 spacer at 20% speed. The resulting milled granulation yieldwas 80.1%. The extragranular excipients were adjusted and bag blendedtogether. The resulting final blend was compressed using a gravity feedframe and hopper at approximated 42 rpm on a Korsch PHI 03 tablet pressequipped with 0.2000×0.4000 oval plain (Hob#21971) tablet tooling forthe 100 mg strength tablets and 0.3360×0.6720 modified oval (Hob#1907)for the 400 mg strength tablets. A hardness of 10 kp and tablet weigh of175 mg was targeted for the 100 mg strength tablets using Formulation D(larger API particle size) and a hardness of 15 kp and 700 mg weight wastargeted for the 400 mg strength tablets using Formulation D (larger APIparticle size).

Evaluation of 100 mg and 400 mg Strength Tablets Using Formulation D(Larger API Particle Size)

The physical properties of the final blend were compared againstprevious batches of Formulation D (Table 13). The bulk density and flowproperties as measured by compressibility and angle of repose were foundto be very similar. Tap density of the final blend was found to besomewhat less than previous batches and the amount of fines in the sieveanalysis was found to be substantially higher than in the previousformulation granulations utilizing 25% granulating fluid even though thegranulation utilized 28% granulating fluid. The larger sized APIappeared to affect the granulation properties. However, the final blendbehaved well on the tablet press producing consistent tablets that metthe tablet weight and hardness targets with low friability for both the100 mg and 400 mg strength tablets. The tablet properties are presentedin Table 13. Assay testing was performed on the 100 mg strength tabletsand similar to all the previous formulations the potency was high(97.7%) the related substances were low (1.03% total). The dissolutiontesting on both the 100 mg and 400 mg strength tablets indicated a rapidrate of release that easily meets the criteria for an immediate releasetablet. The dissolution profile is presented in Table 23.

TABLE 23 Dissolution of 100 mg and 400 mg Strength Tablets usingFormulation D (Larger API Particle Size) % Dissolved vs Time (min)Tablets Sample 0 10 20 30 45 60 75 Formulation D (larger API particle 045 83 90 93 94 95 size) 100 mg strength tablets Formulation D (largerAPI particle 0 55 83 90 92 93 94 size) 400 mg strength tabletsFormulation F 400 mg strength 0 51 86 95 99 100 102 tablets

Accordingly, the use of the larger particle sized API did not negativelyaffect the dissolution of the tablets or their manufacture. Thedissolution profile of the Formulation D 400 mg strength tablets usingthe larger particle sized API was nearly identical to the stabilitybatch Formulation F. However, the larger particle size did require theuse of more granulation fluid in the manufacturing process.

Example 12

Utilizing the procedures described in examples 6 through 11, the tabletsand capsules may contain the compound of formula I in amounts from about10 mg to about 800 mg. The compound of formula I and inert ingredientsare present in the described percentage amounts by weight, which arereadily determined by one of ordinary skill in the art from the previousexamples. For example, one of ordinary skill in the art, following theprocedures of examples 1 through 12 in an analogous manner, can preparetablets and capsules, in addition to those already set forth, whereinthe compound of formula I is present in amounts of about 10 mg, about 20mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg,about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg,about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg,about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg,about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg,about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg,about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg,about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg,about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg,about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg,about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg,about 580 mg, about 590 mg, about 600 mg, about 610 mg, about 620 mg,about 630 mg, about 640 mg, about 650 mg, about 660 mg, about 670 mg,about 680 mg, about 690 mg, about 700 mg, about 710 mg, about 720 mg,about 730 mg, about 740 mg, about 750 mg, about 760 mg, about 770 mg,about 780 mg, about 790 mg, or about 800 mg.

Example 13

The Phase IIa trial started with a 3-4 week placebo run-in period. 141qualified stage I and II hypertensive patients were randomized to takeeither the compound of formula I 200 mg, 500 mg, or placebo, daily for 4weeks. Key efficacy endpoints of mean 24 hour systolic ambulatory bloodpressure monitoring (ABPM), mean 24 hour diastolic ABPM, mean seatedoffice systolic blood pressure (SBP) and diastolic blood pressure (DBP),mean daytime SBP and DBP, mean nighttime SBP and DBP, and SBP and DBPduring final 2 hours of dosing were assessed. 93 patients (efficacypopulation) were available for ABPM analysis.

The compound of formula I at either 200 mg or 500 mg once-daily produceda statistically significantly greater reduction in systolic anddiastolic blood pressure in stage I and II hypertensive patientscompared to placebo (Table 24). The drug was shown to be safe and welltolerated by patients. Most of the adverse events reported were mild ormoderate in severity and included headaches and minor musculoskeletaland respiratory complaints (Table 25). There were no notable changesobserved in liver function and hematology tests (Table 26).

TABLE 24 Efficacy Endpoints at Week 4 (End of Treatment) Mean 24 hr ABPMMean Seated Office Change (mmHg) Change (mmHg) DBP SBP DBP SBP Thecompound  −9.3 ^(a) −12.2 ^(a) −10.5^(b ) −16.9^(b) of formula I 200 mg(n = 35) The compound −10.1 ^(a) −14.8 ^(a)  −9.8^(b) −17.3^(b) offormula I 500 mg (n = 33) Placebo (n = 25) +0.3  −0.4  +1.6  −4.2 ^(a) P< 0.001 for each dose of the compound of formula I vs. placebo. ^(b)P <0.001 for each dose of the compound of formula I vs. placebo.

TABLE 25 Phase IIa Adverse Events (AE) Placebo The compound of formula IN = 36 200 mg (N = 39) 500 mg (N = 38) n (%) n (%) n (%) At least one AE 9 (25.0)  8 (20.5) 10 (26.3) Musculoskeletal  4 (11.1) 1 (2.6) 2 (5.3)Peripheral Edema 1 (2.8) 0 1 (2.6) Tachycardia 1 (2.8) 0 0 Headache 3(8.3) 2 (5.1) 1 (2.6) Confusion/anxiety/ 3 (8.3) 0 0 nervousnessRespiratory 2 (5.6) 0 3 (7.9) Nausea/gastritis 1 (2.8) 2 (5.1) 1 (2.6)Eczema 0 1 (2.6) 0

TABLE 26 Selected Laboratory Parameters The Compound of formula IPlacebo 200 mg 500 mg (N = 36) (N = 39) (N = 38) Hematology Hb (g/dL)<+0.1 −0.5 −0.7 Mean Change from baseline Ht (%) +0.3 −1.7 −2.2 Meanchange from baseline Liver Functions ALT (U/L) +2.9 −1.5 −0.9 Meanchange from baseline AST (U/L) +1.9 −0.9 −1.2 Mean change from baselineγGT (U/L) +3.4 +0.3 +0.8 Mean change from baseline

Example 14

The Phase IIb trial started with a 4-week placebo run-in period and 261qualified patients with SBP/DBP within 140-179/90-109 mmHg ranges wererandomized to either the compound of formula I 200 mg, 400 mg, 800 mg,irbesartan 300 mg, or placebo, taken daily in the morning for 12 weeks.Key efficacy endpoints of seated SBP, DBP and % of patients with BPcontrol (<140/90 mmHg), and all standard safety parameters wereassessed. Results showed that each dose of the compound of formula Iexhibited statistically dose-dependent greater BP decrease in SBP andDBP (p<0.001), and in BP control (p<0.0013) than placebo. Efficacy ofthe compound of formula I 200 mg was similar to irbesartan, 400 mgachieved better BP control than irbesartan (p<0.05) and 800 mg wasstatistically superior to irbesartan in all endpoints as demonstrated onTable 27. The onset of action of the compound of formula I was rapidwithin 4 hours and the efficacy was sustained over 12 weeks.

TABLE 27 Efficacy Endpoints at Week 12 (End of Treatment) Mean SBP MeanDBP % Pts Change Change Achieving Goal (mmHg) (mmHg) (<140/90 mmHg) Thecompound of −13.2 −7.2 36.2% formula I 200 mg (n = 58) The compound of−14.2 −9.2   51.9% ^(b) formula I 400 mg (n = 58) The compound of  −23.4 ^(a)  −14.3 ^(a)   61.5% ^(a) formula I 800 mg (n = 28)Irbesartan 300 mg (n = 58) −10.7 −7.1 31.5% Placebo (n = 59)     1.8^(b)   0.2 ^(b)   9.3% ^(c) ^(a) P < 0.05 vs. irbesartan. ^(b) P < 0.001for each dose of the compound of formula I vs. placebo, ^(c) P = 0.0013for the compound of formula I 200 mg vs. placebo; P < 0.001 for thecompound of formula I 400 mg vs. placebo and 800 mg vs. placebo each.

No serious adverse events (SAES) were reported during active treatment(Table 28). There were no notable changes observed in liver functiontests (Table 29), creatinine, urea nitrogen and vital signs. Inconclusion, the compound of formula I is a novel, dual mechanism ofaction, extremely potent agent that promises to be an importanttreatment of diabetic nephropathy, chronic or persistently elevatedblood pressure, hypertension and other diseases.

TABLE 28 Phase IIb Most Frequent (≥5%) AEs During Treatment, n (%) TheCompound of formula I Irbesartan Preferred Term/ Placebo 200 mg 400 mg800 mg 300 mg SOC Abbrev. (n = 59) (n = 58) (n = 58) (n = 28) (n = 58)Headache/Nerv 10 (16.9) 0 3 (5.2) 2 (7.1) 4 (6.9) Odema 1 (1.7) 2 (3.4)4 (6.9)  3 (10.7) 2 (3.4) peripheral/ Genrl Dizziness/Nerv 2 (3.4) 0 2(3.4)  3 (10.7) 2 (3.4) Fatigue/Genrl 1 (1.7) 0 4 (6.9) 0 3 (5.2)Migraine/Gastr 3 (5.1) 0 2 (3.4) 0 2 (3.4) Diarrhea/Gastr 0 1 (1.7) 1(1.7) 2 (7.1) 1 (1.7) Abdominal pain/ 1 (1.7) 1 (1.7) 0 2 (7.1) 0 GastrUrinary tract 3 (5.1) 0 0 1 (3.6) 0 infection/Infec

TABLE 29 Phase IIb: Liver Function Tests The Compound of formula IIrbesartan Placebo 200 mg 400 mg 800 mg 300 mg ALT (U/L) 2.4 −0.4 −3.2−7.3 0.2 Mean change from baseline AST (U/L) 1.7 −1.2 −1.5 −5.4 0.1 Meanchange from baseline

Example 15

The study starts with qualified patients with diabetic nephrophathy.Screening assessments and evaluations may be conducted over a period ofno more than 2 weeks. Following screening, all eligible patients willundergo a 4-week placebo run-in period to ensure that baseline bloodpressure remains stable and continues to meet eligibility criteria forrandomization. Eligible patients are randomized to instruct to eitherthe compound of formula I: 200 mg, 400 mg, or 800 mg; irbesartan 300 mg;or placebo, taken daily for 12 weeks. Key efficacy endpoints of seatedsystolic BP, diastolic BP and % of patients with BP control (<140/90mmHg), and all standard safety parameters are assessed. Changes frombaseline to final measurement in the course of treatment are recorded.

It is expected that treatment by each dose of the compound of formula Iof a human subject, for example a human subject having diabeticnephrophathy, will exhibits statistically dose-dependent greater BPdecrease in SBP and DBP, and in BP control than placebo and irbesartan.

What is claimed is:
 1. A method of treating a disorder selected from thegroup consisting of glomerulosclerosis and IGA nephropathy, comprisingadministering to a subject in need thereof an effective amount of acompound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein the amount of thecompound of Formula I, or pharmaceutically acceptable salt thereof,administered to the subject is from 200 mg/day to 800 mg/day.
 2. Themethod according to claim 1, wherein the amount of the compound ofFormula I, or pharmaceutically acceptable salt thereof, administered tothe subject is 200 mg/day, 400 mg/day or 800 mg/day.
 3. The methodaccording to claim 1, wherein the amount of the compound of Formula I,or pharmaceutically acceptable salt thereof, administered to the subjectis 200 mg/day.
 4. The method according to claim 1, wherein the amount ofthe compound of Formula I, or pharmaceutically acceptable salt thereof,administered to the subject is 400 mg/day.
 5. The method according toclaim 1, wherein the amount of the compound of Formula I, orpharmaceutically acceptable salt thereof, administered to the subject is800 mg/day.
 6. The method according to claim 1, wherein the disorder isglomerulosclerosis.
 7. The method according to claim 1, wherein thedisorder is IGA nephropathy.
 8. The method according to claim 7, whereinthe amount of the compound of Formula I, or pharmaceutically acceptablesalt thereof, administered to the subject is 200 mg/day, 400 mg/day or800 mg/day.
 9. The method according to claim 7, wherein the amount ofthe compound of Formula I, or pharmaceutically acceptable salt thereof,administered to the subject is 200 mg/day.
 10. The method according toclaim 7, wherein the amount of the compound of Formula I, orpharmaceutically acceptable salt thereof, administered to the subject is400 mg/day.
 11. The method according to claim 7, wherein the amount ofthe compound of Formula I, or pharmaceutically acceptable salt thereof,administered to the subject is 800 mg/day.